Small molecules for the modulation of mcl-1 and methods of modulating cell death, cell division, cell differentiation and methods of treating disorders

ABSTRACT

This invention relates to compounds which selectively bind to the survival protein MCL-1 with high affinity and selectivity, pharmaceutical compositions containing such compounds and the use of those compounds or compositions for modulating MCL-1 activity and for treating hyperproliferative disorders, angiogenesis disorders, cell cycle regulation disorders, autophagy regulation disorders, inflammatory disorders, and/or infectious disorders and/or for enhancing cellular engraftment and/or wound repair, as a sole agent or in combination with other active ingredients.

The present application claims the benefit of U.S. provisionalapplication No. 61/299,803, filed Jan. 29, 2010, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Funding for the present invention was provided in part by support fromNational Institutes of Heath under contract No. 5P01CA92625.Accordingly, the Government of the United States may have certain rightsin and to the invention

BACKGROUND OF THE INVENTION

Beginning with the discovery of BCL-2 at the t14; 18 chromosomalbreakpoint of follicular lymphoma¹⁻³, the anti-apoptotic members of theBCL-2 family have emerged as key pathogenic proteins in human diseasescharacterized by unchecked cellular survival, such as cancer andautoimmunity. A series of anti-apoptotic proteins including BCL-2,BCL-X_(L), BCL-w, MCL-1, BFL1/A1, and BCL-B promote cellular survival bytrapping the critical apoptosis-inducing BCL-2 homology domain 3 (BH3)α-helix of pro-apoptotic BCL-2 family members⁴. Cancer cells exploitthis physiologic survival mechanism through anti-apoptotic proteinoverexpression, establishing an apoptotic blockade that secures theirimmortality. To overcome this potentially fatal resistance mechanism, apharmacologic quest is underway to develop targeted therapies that bindand block BCL-2 family survival proteins.

Anti-apoptotic proteins contain a hydrophobic binding pocket on theirsurface that engages BH3 α-helices^(4,5). Because Nature's solution toanti-apoptotic targeting involves selective interactions between BH3death domains and anti-apoptotic pockets^(6,7), molecular mimicry of theBH3 α-helix has foamed the basis for developing small moleculemodulators of anti-apoptotic proteins⁸⁻¹⁰. Promising compoundsundergoing clinical evaluation, such as ABT-263¹¹, obatoclax⁹, andAT-101¹², each target three or more anti-apoptotic proteins. Thedevelopment of more precise inhibitors that target individualanti-apoptotic proteins remains a significant challenge due to the oftensubtle differences among BH3-binding pockets. Reminiscent of thelong-term goals in kinase therapeutics, anti-apoptotic inhibitors withgreater specificity would provide finely-tuned therapies to treatdistinct diseases while potentially avoiding unwanted side-effects, suchas those observed for ABT-263¹¹ and AT-101¹³. In addition, suchcompounds would serve as invaluable research tools to dissect thedifferential biological functions of anti-apoptotic proteins.

The specificity of anti-apoptotic proteins for BH3 domains is conferredby the topography of the canonical binding groove and the distinctiveamino acid composition of the interacting BH3 helix. Whereas some BH3domains, such as that of pro-apoptotic BIM, can tightly engage allanti-apoptotic pockets, others are more selective like the BAD BH3 thatbinds BCL-2, BCL-X_(L), and BCL-w and the NOXA BH3 that targets MCL-1and BFL1/A1⁶. The differential binding capacity of BH3 domains and theirmimetics is clinically relevant, as exemplified by the closerelationship between inhibitor binding spectrum and biological activity.For example, ABT-737, the prototype small molecule BH3 mimetic modeledafter the BH3 domain of BAD, was designed to specifically target BCL-2and BCL-X_(L) ¹⁰, and induces apoptosis in select cancers that aredriven by these proteins¹⁴⁻¹⁶. However, ABT-737 fails to show efficacyagainst cancer cells that overexpress MCL-1, as this anti-apoptotic liesoutside the molecule's binding spectrum¹⁵⁻¹⁷. In an effort to overcomethe challenge of designing precision small molecules to selectivelytarget interaction surfaces that are comparatively large and morecomplex, we investigated whether Nature's selective BH3 domains could beused to rapidly identify precise small molecule modulators.

The development of precise inhibitors for discrete anti-apoptotic BCL-2family proteins implicated in pathologic cell survival remains aformidable but pressing challenge. Such compounds would providefinely-tuned molecular probes to study and treat human diseases drivenby specific anti-apoptotic blockades. For example, anti-apoptotic MCL-1has emerged as a major resistance factor in cancer.

MCL-1 overexpression has been linked to the pathogenesis of a variety ofrefractory cancers, including multiple myeloma^(18,19), acute myeloidleukemia¹⁶, melanoma²⁰, and poor prognosis breast cancer²¹. MCL-1 exertsits pro-survival activity at the mitochondrial outermembrane where itneutralizes pro-apoptotic proteins such as NOXA, PUMA, BIM, and BAK. Thecritical role of MCL-1 in selective apoptotic resistance has beenhighlighted by the sensitizing effects of small interfering RNAs thatdownregulate MCL-1 protein levels^(22,23).

Despite the formidable challenges associated with developing precisesmall molecule modulators of biomedically relevant protein targets, theidentification of novel and selective small molecules modulators ofMCL-1 is described herein.

BRIEF SUMMARY OF THE INVENTION

One embodiment of this invention encompasses a compound of the formula:

A)

Wherein:

-   -   X is ═O or —O—R³;    -   R¹ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        —SO₂—R^(2a);    -   R² is hydrogen, halogen, —O—R^(2a), —NH—R^(2a), —S—CN,        —S—R^(2a), —S—CH²—R^(2a) or —SO₂—R^(2a);    -   R^(2a) is C₁-C₈ alkyl, substituted or unsubstituted aryl,        substituted or unsubstituted heteroaryl, substituted or        unsubstituted cycloalkyl, or substituted or unsubstituted        heterocycloalkyl;    -   and R³ is hydrogen, C₁-C₈ alkyl substituted or unsubstituted        aryl, substituted or unsubstituted heteroaryl, substituted or        unsubstituted cycloalkyl, or substituted or unsubstituted        heterocycloalkyl; or

B)

wherein:

-   -   R⁴ and R⁵ are independently hydrogen, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted cycloalkyl, or substituted or        unsubstituted heterocycloalkyl or are taken together to form a        5-7 membered, optionally substituted, heteroaryl or        heterocycloalkyl, provided that no more than one of R⁴ and R⁵ is        hydrogen;    -   And R⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl; or

C)

-   -   Wherein R⁷ is CH₂—CO—R^(7a); —CH₂—NH—SO₂—R^(7a);    -   and R^(7a) is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl; or

D)

-   -   Wherein R⁸ is —CO—R^(8a); —CO—NH—(CH₂), —R^(8a); —CO—CH₂—R^(8a),        —NH—CO—NH—R^(8a); —NH—SO₂—NH—R⁸″; or —NH—CO—R^(8a);        —NH—SO₂—R^(8a)    -   and R^(8a) is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl; or

E)

-   -   wherein R⁹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkeynyl, C₂-C₆        alkynyl, —NH₂, —NHR^(9a),    -   —NR^(9a)R^(9b), —COH, —COR^(9a), —COOH, or —COOR^(9a);    -   R^(9a) and R^(9b) are independently C₁-C₆ alkyl, C₂-C₆ alkeynyl,        C₂-C₆ alkynyl, substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl    -   W is        -   —R¹⁰,        -   —CO—R¹⁰,        -   —CO—(CH₂)—R¹⁰,        -   —CO—NH—R¹⁰,        -   —CO—NH—(CH₂)_(n)—R¹⁰,        -   —CO—NH—CO—NH—(CH₂)—R¹⁰,        -   —CO—NH—SO—NH—(CH₂)_(n)—R¹⁰,        -   —SO—(CH₂)_(n)—R¹⁰,        -   —SO—NH—(CH₂)—R¹⁰,        -   —SO—NH—CO—NH—(CH₂)_(n)—R¹⁰,        -   —SO—NH—SO—NH—(CH₂)_(n)—R¹⁰, or    -   the group

-   -   wherein * represents the point of attachment;    -   R¹⁰ is Z or substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl;    -   R¹² is Z or hydrogen, C₁-C₆ alkyl or —(CH₂)_(n)—R^(12a),    -   R^(12a) is Z or substituted or unsubstituted aryl, substituted        or unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl; or

F)

wherein

-   -   R¹³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or C₁-C₆        alkoxy,    -   and R¹⁴ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl        wherein R¹⁴ may be further substituted by Z; or

G)

wherein

R¹⁵ is hydrogen, hydroxyl, halogen, —CO—(CH₂)_(n)—R^(15a),—CO—NH—(CH₂)_(n)—R^(15a), —CO—NH—CO—NH—(CH₂)_(n)—R^(15a),—CO—NH—SO—NH—(CH₂)_(n)—R^(15a), NH—CO—(CH₂)_(n)—R^(15a),NH—CO—NH—(CH₂)_(n)—R^(15a), —SO—(CH₂)_(n)—R^(15a),—SO—NH—(CH₂)_(n)—R^(15a), —SO—NH—CO—NH—(CH₂)_(n)—R^(15a),—SO—NH—SO—NH—(CH₂)_(n)—R^(15a), or —NH—SO₂—R^(15a); alkyl C1-C8-R15a

-   -   R^(15a) is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl;    -   and R¹⁶ is hydrogen, halogen, hydroxyl or substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl,        substituted or unsubstituted cycloalkyl, or substituted or        unsubstituted heterocycloalkyl; or

H)

wherein

-   -   X is O or S;    -   R¹⁷ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl,        wherein R¹⁷ may be further substituted by Z;    -   R¹⁸ is hydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or        C₁-C₆ alkoxy, wherein R¹⁸ may be further substituted by Z;    -   and R¹⁹ is hydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        or C₁-C₆ alkoxy wherein R¹⁹ may be further substituted by Z; or

I)

wherein

-   -   Y¹ is N, NH, CH or CH₂;    -   Y² is O, S, or NH;    -   ---- represents an optional double bond;    -   R²⁰ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl,        wherein R²⁰ may be further substituted by Z;    -   R²¹ represents ═S, ═NH, —NHR^(12a) CHR^(21a), ═NR^(21a), or        ═CCO—R^(21a); and    -   R^(21a) is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl,        wherein R^(21a) may be further substituted by Z; or

J)

wherein

-   -   R²² is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl        wherein R²² may be further substituted by Z;    -   R²³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or C₁-C₆        alkoxy, substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl        wherein R²³ may be further substituted by Z; and    -   R²⁴ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or C₁-C₆        alkoxy, substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl        wherein R²⁴ may be further substituted by Z; or

K)

wherein

-   -   R²⁵ is —CO—R^(25a); —CO—NH—(CH₂)_(n)—R^(25a); —CO—CH₂—R^(25a),        —NH—CO—NH—R^(25a); —NH—SO₂—NH—R^(25a); or —NH—CO—R^(25a);        —NH—SO₂—R^(25a)    -   R^(25a) is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl        wherein R²² may be further substituted by Z; or

L)

wherein

-   -   R²⁶ is substituted or unsubstituted aryl, substituted or        unsubstituted heteroaryl, substituted or unsubstituted        cycloalkyl, or substituted or unsubstituted heterocycloalkyl        wherein R²² may be further substituted by Z; or        -   and wherein        -   in each of formulas A-L:        -   each occurrence of n is independently an integer from 0-4;        -   each occurrence of m is independently an integer from 0-3;        -   each occurrence of p is independently an integer from 0-2;        -   each occurrence of q is independently an integer from 0-1;        -   each occurrence of Z, Z′ and Z″ is independently halogen,            hydroxyl, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or            C₁-C₆ alkoxy, substituted or unsubstituted aryl, substituted            or unsubstituted heteroaryl, substituted or unsubstituted            cycloalkyl, or substituted or unsubstituted            heterocycloalkyl,

or M)

-   -   a compound having the formula

T¹L-T²_(u)Q-T²

wherein:

-   -   T¹ represents a compound of the formula A-L;    -   each T² independently represents another compound of the formula        A-L which may be the same or different from T¹;    -   each Q represents a direct bond, a C₁-C₈ alkylene linker, a        C₂-C₈ alkenylene linker, or a C₂-C₈ alkynylene linker, an amide        linker, or a sulfonamide linker formed by joining together a Z,        Z′, or Z″ moiety of T¹ with a Z, Z′, or Z″ moiety of each        subsequent T²; and    -   u represents an integer from 0-5    -   or a pharmaceutically acceptable salt, solvate, hydrate or        stereoisomer thereof.

In some embodiments, the invention encompasses a compound of having theformula M:

T¹L-T²_(u)Q-T²

wherein:

-   -   T¹ represents a compound of the formula A-L;    -   each T² independently represents another compound of the formula        A-L which may be the same or different from T¹;    -   each Q represents a direct bond, a C₁-C₈ alkylene linker, a        C₂-C₈ alkenylene linker, or a C₂-C₈ alkynylene linker formed by        joining together a Z, Z′, or Z″ moiety of T¹ with a Z, Z′, or Z″        moiety of each subsequent T²; and    -   u represents an integer from 0-5    -   or a pharmaceutically acceptable salt, solvate, hydrate or        stereoisomer thereof.

In some embodiments, the invention encompasses a compound having theformula M wherein u is 0 and Q is a direct bond.

In still other embodiments, the invention encompasses a compound havingthe formula M wherein u is 0 and Q is a C₁-C₈ alkylene linker.

In yet other embodiments, the invention encompasses a compound havingthe formula M wherein u is 1 and each Q is independently a direct bondor a C₁-C₈ alkylene linker.

Representative compounds and subclasses of compounds of the inventionare identified in Table 1 (including Subtables Table 1-A through Table1-P). These compounds may be useful in the methods and compositionsdescribed herein.

Another aspect of the invention encompasses a pharmaceutical compositioncomprising one or more compounds according to Formula A-M orphysiologically acceptable salts, solvates, hydrates, stereoisomers, orfusion compounds thereof and a pharmaceutically acceptable diluent orcarrier. In some embodiments, the pharmaceutical composition theinvention encompasses a composition wherein the compound is present in atherapeutically effective amount.

In still another embodiment, the pharmaceutical composition theinvention further encompasses at least one further active compound. Inone embodiment, the further active compound is a death receptorstimulant, an HDAC inhibitor, a proteasome inhibitor, a BCL-2 familyinhibitor, a kinase inhibitor, a mitotic inhibitor, a nucleoside analog,an anti-cancer monoclonal antibody, a corticosteroid, a DNA-damagingagent, an antimetabolite, or other cell death-activating stimulant.

Another aspect of the invention encompasses a packaged pharmaceuticalcomposition comprising a container, the pharmaceutical composition ofthe invention and instructions for using the pharmaceutical compositionto treat a disease or condition in a mammal.

Yet another aspect of the invention encompasses a method of modulatingMCL-1 in a cell comprising contacting a cell with one or more compoundsof the invention. In one embodiment, the activity modulated is apoptoticcell death. In another embodiment, the activity modulated is autophagy.In another embodiment, the activity modulated is necrotic cell death. Inanother embodiment, the activity modulated is cell metabolism. Inanother embodiment, the activity modulated is cell division. In anotherembodiment, the activity modulated is transcription in a cell. Inanother embodiment, the activity modulated is RNA Processing in a cell.In another embodiment, the activity modulated is cell differentiation.In another embodiment, the activity modulated is transcription. Inanother embodiment, the activity modulated is RNA processing. In anotherembodiment, the activity modulated is protein multimerization ordissociation.

Still another aspect of the invention encompasses a method of treating ahyperproliferative disorder in a mammal comprising administering to amammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention. In some embodiments, thehyperproliferative disorder is cancer, including but not limited to,cancer of the breast, respiratory tract, brain, reproductive organs,digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid or a distant metastasis of a solid tumor, alymphoma, sarcoma, melanoma or leukemia.

Yet another aspect of the invention encompasses a method of treating anangiogenesis disorder in a mammal comprising administering to a mammalin need thereof, a therapeutically effective amount of one or morecompounds of the invention.

Still yet another aspect of the invention encompasses a method oftreating an inflammatory disorder in a mammal comprising administeringto a mammal in need thereof, a therapeutically effective amount of oneor more compounds of the invention.

Another aspect of the invention encompasses a method of treating aninfectious disease in a mammal comprising administering to a mammal inneed thereof, a therapeutically effective amount of one or morecompounds of the invention.

Still another aspect of the invention encompasses a method of treating acell cycle regulation disorder in a mammal comprising administering to amammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention.

Yet another aspect of the invention encompasses a method of treating anautophagy regulation disorder in a mammal comprising administering to amammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention.

Still yet another aspect of the invention encompasses a method oftreating an autoimmune disorder in a mammal comprising administering toa mammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention.

Another aspect of the invention encompasses a method of enhancing woundrepair in a mammal comprising administering to a mammal in need thereof,a therapeutically effective amount of one or more compounds of theinvention.

Yet another aspect of the invention encompasses a method of enhancingcellular engraftment in a mammal comprising administering to a mammal inneed thereof, a therapeutically effective amount of one or morecompounds of the invention.

Another aspect of the invention encompasses a method of sensitizing acell to treatment by a secondary active ingredient comprisingadministering to a cell a sensitizing amount of one or more compounds ofthe invention. In certain embodiments, the secondary active ingredientis a death receptor stimulant, an RDAC inhibitor, a proteasomeinhibitor, a BCL-2 family inhibitor, a kinase inhibitor, a mitoticinhibitor, a nucleoside analog, an anti-cancer monoclonal antibody, acorticosteroid, a DNA-damaging agent, an antimetabolite, or other celldeath-activating stimulant.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows the application of a selective stapled peptide in acompetitive binding screen to identify selective small molecules againsta protein target.

FIG. 2A shows a surface view of the BH3-binding pocket of MCL-1ΔNΔC fromthe X-ray structure of MCL-1 (PDB ID: 2PQK), with discrete topographicregions of the binding surface labeled A-E.

FIG. 2B shows examples of selective small molecule binders of MCL-1ΔNΔCengaging discrete topographic regions (labeled A-E) of the BH3-bindingpocket, as rendered by molecular docking. Since certain small moleculescover distinct regions of the pocket, synthetic combinations ofmolecules or fragments thereof can yield a larger molecule thatselectively binds a greater surface area of the BH3-binding pocket.

FIG. 2C shows the results of a dilutional assay that revealed selectivesmall molecule binders can be subclassified based on engaging MCL-1through non-covalent or covalent interactions.

FIG. 2D shows that a subgroup of MCL-1 inhibitor molecules covalentlyreacted with C286 of MCL-1 at a novel interaction site.

FIG. 2E demonstrates that C286S mutagenesis abrogates binding of thissubgroup of molecules, continuing engagement of a novel interaction sitefor MCL-1 inhibition.

FIG. 3 shows competitive fluorescence polarization binding assays (A)and tabulated 1050 values (B) demonstrating that small moleculebioactives identified as MCL-1 selective indeed exhibit preferentialdisplacement of FITC-BID BH3 from MCL-1ΔNΔC compared to otheranti-apoptotic proteins.

FIG. 4 shows competitive fluorescence polarization binding assays (A)and tabulated IC50 values (B) demonstrating that small moleculesidentified as MCL-1 selective exhibit preferential displacement ofFITC-BID BH3 from MCL-1ΔNΔC compared to other anti-apoptotic proteins.Structure-activity relationship (SAR) analysis for select derivatives of(C) Class A and (D) Class J molecular inhibitors of MCL-1, using theFITC-BID BH3/MCL-1ΔNΔC competitive fluorescence polarization bindingassay.

FIG. 5A shows that a SAHB peptide selective for MCL-1 sensitizesTRAIL-induced and caspase-dependent cell death of Jurkat T-cell leukemiaand OPM2 multiple myeloma cells.

FIG. 5B shows that small molecule bioactives that target MCL-1 alsosensitize the capacity of TRAIL to inhibit viability of OPM2 cells, ashas been shown for selective targeting of MCL-1 with MCL-1 SAHB (FIG.5A) and for selective knock-down of MCL-1 with siRNA.

FIG. 5C shows a combination treatment analysis using CalcuSyn softwarerevealing combination index values that reflect synergy betweentreatment with TRAIL and the identified small molecule bioactive hitsthat target MCL-1.

FIG. 5D shows a combination treatment analysis using CalcuSyn softwarerevealing combination index values that reflect synergy betweentreatment with TRAIL and the identified small molecule hits that targetMCL-1.

FIG. 6 shows that selective MCL-1 inhibitor molecules block theanti-apoptotic activity of MCL-1 in a BAX-mediated liposomal releaseassay.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In order that the invention may be more readily understood, certainterms are first defined and collected here for convenience. Otherdefinitions appear in context throughout the application.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The term “halogen” refers to radicals of fluorine, chlorine, bromine andiodine.

The term “alkyl” refers to a straight or branched hydrocarbon chainradical, containing solely carbon and hydrogen atoms, having in therange from one up to eight carbon atoms, and which is attached to therest of the molecule by a single bond, such as illustratively, methyl,ethyl, n-propyl 1-methylethyl (iso-propyl), n-butyl, n-pentyl, and1,1-dimethylethyl (tert-butyl).

The term “cycloalkyl” denotes a non-aromatic mono or multicyclic ringsystem having in the range of 3 up to 14 carbon atoms such ascyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples ofmulticyclic cycloalkyl groups include decahydronapththyl. Examples ofbridged cycloalkyl groups or sprirobicycloalkyl groups include adamantylnorbornyl, and sprio[4.4]nonyl groups.

The term “alkoxy” denotes an alkyl group as defined herein attached viaan oxygen linkage to the rest of the molecule. Representative examplesof those groups are methoxy, ethoxy, iso-propoxy, n-butoxy, andtert-butoxy.

The term “cycloalkoxy” denotes a cycloalkyl group as defined hereinattached via an oxygen linkage to the rest of the molecule.Representative examples of those groups are cyclopropoxy, cyclobutoxy,cyclopentoxy, cyclohexoxy, and cycloheptoxy.

The term “aryl” refers to aromatic radicals having in the range of 6 upto 14 carbon atoms such as phenyl, naphthyl, indanyl, and biphenyl.

The term “heteroaryl” refers to a stable 5- to 13-membered aromaticheterocycle having in the range of from 1 up to 4 heteroatoms from thegroup consisting of nitrogen, phosphorus, oxygen and sulfur, which ringor ring system can be linked via a carbon atom or a nitrogen atom, ifsuch an atom is present. For purposes of this invention, the heteroarylring radical may be a monocyclic, bicyclic or tricyclic ring system.Examples of such heteroaryl radicals are: pyridyl, pyridyl N-oxide,pyrimidyl, pyridazinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl or isoxazolyl, indolicenyl, indolyl,benzo[b]thienyl, benzo[b]furyl, benzothiazolyl, benzothiadiazolyl,indazolyl, quinolyl, isoquinolyl, isoquinolyl, naphthyridinyl,quinazolinyl, oxadiazolyl, benzoxazolyl, tetrazoyl, triazolyl,thiadiazolyl, and benzimidazolyl.

The term “heterocycloalkyl” refers to a stable 3 to 13 memberedsaturated or partially unsaturated heterocycle having in the range from1 up to 4 heteroatoms from the group consisting of nitrogen, phosphorus,oxygen and sulfur, which ring or ring system can be linked via a carbonatom or a nitrogen atom, if such an atom is present. For purposes ofthis invention, the heterocyclic ring radical may be a monocyclic,bicyclic or tricyclic ring system, which may include fused, bridged orspiro ring systems. Examples of such heterocyclyl radicals are:tetrahydropyranyl, aziridyl, azepanyl, tetrahydrofuryl, pyrrolidinyl,pyrrolinyl, piperidinyl, 1,2 dihydropyridinyl, 1,4 dihydropyridinyl,piperazinyl, morpholinyl, thiomorpholinyl, azepinyl, oxazolinyl,thiazolinyl and 1,4 diazepinyl.

The term “alkylamino” refers to an alkyl group as defined hereinattached via amino linkage to the rest of the molecule. The termalkylamino further includes dialkyl amino moieties in which two alkylgroups as define herein are attached via amino linkage to the rest ofthe molecule. Representative examples of those groups are methylaminoand dimethylamino.

Where the plural form of the word compounds, salts, polymorphs,hydrates, solvates and the like, is used herein, this is taken to meanalso a single compound, salt, polymorph, isomer, hydrate, solvate or thelike.

The compounds of this invention may contain one or more asymmetriccenters, depending upon the location and nature of the varioussubstituents desired. Asymmetric carbon atoms may be present in the (R)or (S) configuration, resulting in racemic mixtures in the case of asingle asymmetric center, and diastereomeric mixtures in the case ofmultiple asymmetric centers. In certain instances, asymmetry may also bepresent due to restricted rotation about a given bond, for example, thecentral bond adjoining two substituted aromatic rings of the specifiedcompounds. Substituents on a ring may also be present in either cis ortrans form. It is intended that all such configurations (includingenantiomers and diastereomers), are included within the scope of thepresent invention. Preferred compounds are those which produce the moredesirable biological activity. Separated, pure or partially purifiedisomers and stereoisomers or racemic or diastereomeric mixtures of thecompounds of this invention are also included within the scope of thepresent invention. The purification and the separation of such materialscan be accomplished by standard techniques known in the art.

The optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes, for example, by theformation of diastereoisomeric salts using an optically active acid orbase or formation of covalent diastereomers. Examples of appropriateacids are tartaric, diacetyltartaric, ditoluoyltartaric andcamphorsulfonic acid. Mixtures of diastereoisomers can be separated intotheir individual diastereomers on the basis of their physical and/orchemical differences by methods known in the art, for example, bychromatography or fractional crystallization. The optically active basesor acids are then liberated from the separated diastereomeric salts. Adifferent process for separation of optical isomers involves the use ofchiral chromatography (e.g., chiral HPLC columns), with or withoutconventional derivitization, optimally chosen to maximize the separationof the enantiomers. Suitable chiral HPLC columns are manufactured byDiacel, e.g., Chiracel OD and Chiracel OJ among many others, allroutinely selectable. Enzymatic separations, with or withoutderivitization, are also useful. The optically active compounds of thisinvention can likewise be obtained by chiral syntheses utilizingoptically active starting materials.

The present invention also relates to useful forms of the compounds asdisclosed herein, such as pharmaceutically acceptable salts,co-precipitates, metabolites, hydrates, solvates and prodrugs of all thecompounds of examples. The term “pharmaceutically acceptable salt”refers to a relatively non-toxic, inorganic or organic acid additionsalt of a compound of the present invention. For example, see S. M.Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci. 1977, 66, 1-19.Pharmaceutically acceptable salts include those obtained by reacting themain compound, functioning as a base, with an inorganic or organic acidto form a salt, for example, salts of hydrochloric acid, sulfuric acid,phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalicacid, maleic acid, succinic acid and citric acid.

Pharmaceutically acceptable salts also include those in which a compoundof the invention functions as an acid and is reacted with an appropriatebase to form, e.g., sodium, potassium, calcium, magnesium, ammonium, andchorine salts. Those skilled in the art will further recognize that acidaddition salts of the claimed compounds may be prepared by reaction of acompound of the invention the appropriate inorganic or organic acid viaany of a number of known methods. Alternatively, alkali and alkalineearth metal salts of acidic compounds of the invention are prepared byreacting the compounds of the invention with the appropriate base via avariety of known methods.

Representative salts of a compound of the invention include theconventional non-toxic salts and the quaternary ammonium salts which areformed, for example, from inorganic or organic acids or bases by meanswell known in the art. For example, such acid addition salts includeacetate, adipate, alginate, ascorbate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cinnamate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate,maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate,3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfonate,tartrate, thiocyanate, tosylate, and undecanoate.

Base salts include alkali metal salts such as potassium and sodiumsalts, alkaline earth metal salts such as calcium and magnesium salts,and ammonium salts with organic bases such as dicyclohexylamine andN-methyl-D-glucamine. Additionally, basic nitrogen containing groups maybe quaternized with such agents as lower alkyl halides such as methyl,ethyl, propyl, and butyl chlorides, bromides and iodides; dialkylsulfates like dimethyl, diethyl, and dibutyl sulfate; and diamylsulfates, long chain halides such as decyl, lauryl, myristyl and stearylchlorides, bromides and iodides, aralkyl halides like benzyl andphenethyl bromides and others.

A solvate for the purpose of this invention is a complex of a solventand a compound of the invention in the solid state. Exemplary solvateswould include, but are not limited to, complexes of a compound of theinvention with ethanol or methanol. Hydrates are a specific form ofsolvate wherein the solvent is water.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In certain embodiments, thesubject is a human.

It is also to be understood that the terminology used herein is forpurposes of describing particular embodiments only, and is not intendedto be limiting. As used in the specification and the appended claims,the singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly indicates otherwise. Thus, for example, reference to“a peptide” includes multiple peptides, reference to “a spacer” includestwo or more spacers.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference.

Compounds

As set forth above, the invention provides compounds which selectivelybind to the survival protein MCL-1 with high affinity and selectivity.

Table 1 (including Subtables Table 1-A through Table 1-P) lists ofrepresentative compounds and subclasses identified as selective orpreferential MCL-1 binders based upon a small molecule competitivebinding assay using FITC-MCL-1 SAHB/MCL-1ΔNΔC and FITC-BADBH3/BCL-X_(L)ΔC interaction pairs. FIGS. 4C and 4D tabulates selectderivatives of Class A and Class J compounds that exhibit differentialMCL-1 targeting activity based on chemical variation of discrete Rgroups.

Certain compounds of Table 1 describe certain known bioactive compounds.These compounds are particularly useful in targeting MCL-1 and may beuseful in the treatment of diseases as described herein. Thus, incertain aspects, the compounds of Formula A-L may exclude certaincompounds of Table 1.

The compounds of the invention may contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of these compounds are expressly included in theinvention. The compounds of this invention may also be represented inmultiple tautomeric forms, in such instances, the invention expresslyincludes all tautomeric forms of the compounds described herein. Allsuch isomeric forms of such compounds are expressly included in theinvention. All crystal forms of the compounds described herein areexpressly included in the invention.

Naturally occurring or synthetic isomers can be separated in severalways known in the art. Methods for separating a racemic mixture of twoenantiomers include chromatography using a chiral stationary phase (see,e.g., “Chiral Liquid Chromatography,” W. J. Lough, Ed. Chapman and Hall,New York (1989)). Enantiomers can also be separated by classicalresolution techniques. For example, formation of diastereomeric saltsand fractional crystallization can be used to separate enantiomers. Forthe separation of enantiomers of carboxylic acids, the diastereomericsalts can be formed by addition of enantiomerically pure chiral basessuch as brucine, quinine, ephedrine, strychnine, and the like.Alternatively, diastereomeric esters can be formed with enantiomericallypure chiral alcohols such as menthol, followed by separation of thediastereomeric esters and hydrolysis to yield the free, enantiomericallyenriched carboxylic acid. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

Methods of obtaining a compound of the invention include purchasing,synthesizing or otherwise acquiring the compound. Synthesizing acompound of the invention is within the means of chemists of ordinaryskill in the art. Methods for optimizing reaction conditions, ifnecessary minimizing competing by-products, are known in the art. Themethods may also additionally include steps, either before or after thesteps described specifically herein, to add or remove suitableprotecting groups in order to ultimately allow synthesis of thecompounds herein. In addition, various synthetic steps may be performedin an alternate sequence or order to give the desired compounds.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the applicablecompounds are known in the art and include, for example, those describedin R. Larock, Comprehensive Organic Transformations, VCH Publishers(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995) and subsequent editions thereof.

Methods of Treatment

In one aspect, the invention encompasses a method of modulating MCL-1 ina cell comprising contacting a cell with one or more compounds of theinvention. Such modulation is useful in the modulation of programmedcell death or apoptosis, autophagic cell death; necrotic cell death;cell metabolism; cell division; cell differentiation; cell migration;cell engraftment; tissue repair; RNA transcription and processing; orprotein multimerization or dissociation.

As used herein, the term “modulation” refers to a change in activity asa direct or indirect response to the presence of at least one compoundof the invention described herein, relative to the activity in theabsence of the compound. The change may be an increase in activity or adecrease in activity, and may be due to the direct interaction of thecompound with MCL-1, or due to the interaction of the compound with oneor more other factors that in turn interact with MCL-1 or affect MCL-1activity. In certain embodiments, the modulation may be the result of acovalent bond between the compound with MCL-1. In certain embodiments,the modulation may be the result of a non-covalent bond between thecompound with MCL-1. In certain embodiments, the modulation may be theresult of a covalent bond between the compound with the established BH3binding site of MCL-1 or with a residue of MCL-1 not associated with theestablished BH3 binding site of MCL-1. In certain embodiments, themodulation may be the result of a non-covalent bond between the compoundwith the established BH3 binding site of MCL-1 or with a residue ofMCL-1 not associated with the established BH3 binding site of MCL-1. Instill other embodiments, the modulation may be the result of a covalentbond between the compound with the C286 reside of MCL-1. In certainembodiments, the modulation may be the result of a non-covalent bondbetween the compound in the region of the C286 residue of MCL-1.

In one aspect, the invention encompasses a method of treating ahyperproliferative disorder in a mammal comprising administering to amammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention.

As used herein the term “hyperproliferative disorder” refers to adisorder associated with an irregular or an abnormally high rate of celldivision (which results in a rapid proliferation of the cells) or ablockade in the natural cell death pathway resulting in the accumulationof cells, or a combination thereof. Hyperproliferative disorders includebut are not limited, e.g., psoriasis, keloids, and other hyperplasiasaffecting the skin, benign prostate hyperplasia (BPH), solid and liquidtumors, such as cancers of the breast, respiratory tract, brain,reproductive organs, digestive tract, urinary tract, eye, liver, skin,head and neck, thyroid, parathyroid and their distant metastases,lymphomas, sarcomas, and leukemias.

Still another aspect of the invention encompasses a method of treating acell cycle regulation disorder in a mammal comprising administering to amammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention.

As used herein, the term “cell cycle regulation disorder” relates to adisorder of the cell cycle regulation mechanisms, observed in manymalignant tumors, are directly related to uncontrolled proliferation ofcancer cells. Cell cycle regulation disorders can be the result of thedegradation of the regulation of one or more stages of cell growth, G₁,S, G₂, and M phases of the growth cycle and may include the degradationof one or more cell cycle regulators including, but not limited to,p27Kip1, cdk2, cdk4, cdk6, chk1, cdc25, cyclin B, cyclin H, cyclin D,and cyclin E.

Examples of breast cancer include, but are not limited to invasiveductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ,and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are notlimited to small-cell and non-small-cell lung carcinoma, as well asbronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to brain stem andhypophtalmic glioma, cerebellar and cerebral astrocytoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumor.

Tumors of the male reproductive organs include, but are not limited toprostate and testicular cancer. Tumors of the female reproductive organsinclude, but are not limited to endometrial, cervical, ovarian, vaginal,and vulvar cancer, as well as sarcoma of the uterus.

Tumors of the digestive tract include, but are not limited to anal,colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal,small-intestine, and salivary gland cancers.

Tumors of the urinary tract include, but are not limited to bladder,penile, kidney, renal pelvis, ureter, urethral and human papillary renalcancers.

Eye cancers include, but are not limited to intraocular melanoma andretinoblastoma.

Examples of liver cancers include, but are not limited to hepatocellularcarcinoma (liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to laryngeal,hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oralcavity cancer and squamous cell. Lymphomas include, but are not limitedto AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-celllymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of thecentral nervous system. Sarcomas include, but are not limited to sarcomaof the soft tissue, osteosarcoma, malignant fibrous histiocytoma,lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

In one aspect, the invention encompasses a method of treatingangiogenesis in a mammal comprising administering to a mammal in needthereof, a therapeutically effective amount of one or more compounds ofthe invention.

As used herein the term “angiogenesis” refers to a disorder associatedwith excessive and/or abnormal angiogenesis.

Inappropriate and ectopic expression of angiogenesis can be deleteriousto an organism. A number of pathological conditions are associated withthe growth of extraneous blood vessels. These include, e.g., diabeticretinopathy, ischemic retinal-vein occlusion, and retinopathy ofprematurity (Aiello et al. New Engl. J. Med. 1994, 331, 1480; Peer etal. Lab. Invest. 1995, 72, 638), age-related macular degeneration (AMD;see, Lopez et al. Invest. Opththalmol. Vis. Sci. 1996, 37, 855),neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, rheumatoid arthritis (RA), restenosis, in-stentrestenosis, vascular graft restenosis, etc. In addition, the increasedblood supply associated with cancerous and neoplastic tissue, encouragesgrowth, leading to rapid tumor enlargement and metastasis. Moreover, thegrowth of new blood and lymph vessels in a tumor provides an escaperoute for renegade cells, encouraging metastasis and the consequencespread of the cancer. Thus, compounds of the present invention can beutilized to treat and/or prevent any of the aforementioned angiogenesisdisorders, e.g., by inhibiting and/or reducing blood vessel formation;by inhibiting, blocking, reducing, decreasing, etc. endothelial cellproliferation or other types involved in angiogenesis, as well ascausing cell death or apoptosis of such cell types.

Another aspect of the invention encompasses a method of treating aninfectious disease by blocking pathogen or infected cell survival in amammal comprising administering to a mammal in need thereof, atherapeutically effective amount of one or more compounds of theinvention.

Examples of infectious diseases include, but are not limited to, AIDS,Alveolar Hydatid Disease (AHD, Echinococcosis), Amebiasis (Entamoebahistolytica Infection), Angiostrongylus Infection, Anisakiasis, Anthrax,Babesiosis (Babesia Infection), Balantidium Infection (Balantidiasis),Baylisascaris Infection (Raccoon Roundworm), Bilharzia(Schistosomiasis), Blastocystis hominis Infection (Blastomycosis),Boreliosis, Botulism, Brainerd Diarrhea, Brucellosis, BSE (BovineSpongiform Encephalopathy), Candidiasis, Capillariasis (CapillariaInfection), CFS (Chronic Fatigue Syndrome), Chagas Disease (AmericanTrypanosomiasis), Chickenpox (Varicella-Zoster virus), Chlamydiapneumoniae Infection, Cholera, Chronic Fatigue Syndrome, CJD(Creutzfeldt-Jakob Disease), Clonorchiasis (Clonorchis Infection), CLM(Cutaneous Larva Migrans, Hookworm Infection), Coccidioidomycosis,Conjunctivitis, Coxsackievirus A16 (Hand, Foot and Mouth Disease),Cryptococcosis, Cryptosporidium Infection (Cryptosporidiosis), Culexmosquito (Vector of West Nile Virus), Cutaneous Larva Migrans (CLM),Cyclosporiasis (Cyclospora Infection), Cysticercosis(Neurocysticercosis), Cytomegalovirus Infection, Dengue/Dengue Fever,Dipylidium Infection (Dog and Cat Flea Tapeworm), Ebola VirusHemorrhagic Fever, Echinococcosis (Alveolar Hydatid Disease),Encephalitis, Entamoeba coli Infection, Entamoeba dispar Infection,Entamoeba hartmanni Infection, Entamoeba histolytica Infection(Amebiasis), Entamoeba polecki Infection, Enterobiasis (PinwormInfection), Enterovirus Infection (Non-Polio), Epstein-Barr VirusInfection, Escherichia coli Infection, Foodborne Infection, Foot andmouth Disease, Fungal Dermatitis, Gastroenteritis, Group A streptococcalDisease, Group B streptococcal Disease, Hansen's Disease (Leprosy),Hantavirus Pulmonary Syndrome, Head Lice Infestation (Pediculosis),Heliobacter pylori Infection, Hematologic Disease, Hendra VirusInfection, Hepatitis (HCV, HBV), Herpes Zoster (Shingles), HIVInfection, Human Ehrlichiosis, Human Parainfluenza Virus Infection,Influenza, Isosporiasis (Isospora Infection), Lassa Fever,Leishmaniasis, Kala-azar (Kala-azar, Leishmania Infection), Lice (Bodylice, Head lice, Pubic lice), Lyme Disease, Malaria, Marburg HemorrhagicFever, Measles, Meningitis, Mosquito-borne Diseases, Mycobacterium aviumComplex (MAC) Infection, Naegleria Infection, Nosocomial Infections,Nonpathogenic Intestinal Amebae Infection, Onchocerciasis (RiverBlindness), Opisthorciasis (Opisthorcis Infection), ParvovirusInfection, Plague, PCP (Pneumocystis carinii Pneumonia), Polio, Q Fever,Rabies, Respiratory Syncytial Virus (RSV) Infection, Rheumatic Fever,Rift Valley Fever, River Blindness (Onchocerciasis), RotavirusInfection, Roundworms Infection, Salmonellosis, Salmonella Enteritidis,Scabies, Shigellosis, Shingles, Sleeping Sickness, Smallpox,Streptococcal Infection, Tapeworm Infection (Taenia Infection), Tetanus,Toxic Shock Syndrome, Tuberculosis, Ulcers (Peptic Ulcer Disease),Valley Fever, Vibrio parahaemolyticus Infection, Vibrio vulnificusInfection, Viral Hemorrhagic Fever, Warts, Waterborne infectiousDiseases, West Nile Virus Infection (West Nile Encephalitis), WhoopingCough, Yellow Fever, tuberculosis, leprosy, mycobacteria-inducedmeningitis, Chagas disease, effects of Shiga-like toxin resulting fromStaphylococcus infection, meningococcal infection, and infections fromBorrelia burgdorferi or Treponema pallidum.

Yet another aspect of the invention encompasses a method of treating anautophagy regulation disorder in a mammal comprising administering to amammal in need thereof, a therapeutically effective amount of one ormore compounds of the invention.

As used herein, the term “autophagy regulation disorder” relates to adissorder of the autophagy system of a cell i.e. the self-digestion by acell through the action of enzymes originating within the same cellincluding, but not limited to, chaperone-mediated autophagy,macroautophagy, microautophagy. Disorders of autophagy regulationinclude but are not limited to, Danon disease X-linked myopathy withexcessive autophagy, Infantile autophagic vacuolar myopathy, Childhoodautophagic vacuolar myopathy, Adult-onset autophagic vacuolar myopathywith multiorgan involvement, LGMD 1A, Toxic myopathy, Inflammatorymyopathies, Hypokalemic myopathy, Acid maltase deficiency, Inflammatorybowel disease associations, Autophagy 16-like 1 (ATG16L1), LRG47, andautophagy-mediated survival of cancer cells or cancer stem cells toescape chemotherapy or radiation treatments.

Still yet another aspect of the invention provides a method of treatingan autoimmune disorder in a mammal comprising administering to a mammalin need thereof, a therapeutically effective amount of one or morecompounds of the invention.

Examples of autoimmune disorders include, but are not limited to,allergic bronchopulmonary aspergillosis; autoimmune hemolytic anemia;acanthosis nigricans; allergic contact dermatitis; Addison's disease;atopic dermatitis; alopecia greata; alopecia universalis; amyloidosis;anaphylactoid purpura; anaphylactoid reaction; aplastic anemia;angioedema, hereditary; angioedema, idiopathic; ankylosing spondylitis;arteritis, cranial; arteritis, giant cell; arteritis, Takayasu's;arteritis, temporal; asthma; a-telangiectasia; autoimmune oophoritis;autoimmune orchitis; autoimmune polyendocrine failure; Behcet's disease;Berger's disease; Buerger's disease; bullous pemphigus; candidiasis,chronic mucocutaneous; Caplan's syndrome; post-myocardial infarctionsyndrome; post-pericardiotomy syndrome; carditis; celiac sprue; Chagas'sdisease; Chediak-Higashi syndrome; Churg-Strauss disease; Cogan'ssyndrome; cold agglutinin disease; CREST syndrome; Crohn's disease;cryoglobulinemia; cryptogenic fibrosing alveolitis; herpetifomis;dennatomyositis; diabetes mellitus; Diamond-Blackfan syndrome; DiGeorgesyndrome; discoid lupus erythematosus; eosinophilic fascitis;episcleritis; drythema elevatum diutinum; erythema marginatum; erythemamultiforme; erythema nodosum; familial Mediterranean fever; Felty'ssyndrome; fibrosis pulmonary; glomerulonephritis, anaphylactoid;glomerulonephritis, autoimmune; glomerulonephritis, post-streptococcal;glomerulonephritis, post-transplantation; glomerulopathy, membranous;Goodpasture's syndrome; graft-vs.-host disease; granulocytopenia,immune-mediated; granuloma annulare; granulomatosis, allergic;granulomatous myositis; Grave's disease; Hashimoto's thyroiditis;hemolytic disease of the newborn; hemochromatosis, idiopathic;Henoch-Schoenlein purpura; hepatitis, chronic active and chronicprogressive; histiocytosis X; hypereosinophilic syndrome; idiopathicthrombocytopenic purpura; Job's syndrome; juvenile dermatomyositis;juvenile rheumatoid arthritis juvenile chronic arthritis); Kawasaki'sdisease; keratitis; keratoconjunctivitis sicca;Landry-Guillain-Barre-Strohl syndrome; leprosy, lepromatous; Loeffler'ssyndrome; Lyell's syndrome; Lyme disease; lymphomatoid granulomatosis;mastocytosis, systemic; mixed connective tissue disease; mononeuritismultiplex; Muckle-Wells syndrome; mucocutaneous lymph node syndrome;mucocutaneous lymph node syndrome; multicentric reticulohistiocytosis;multiple sclerosis; myasthenia gravis; mycosis fungoides; necrotizingvasculitis, systemic; nephrotic syndrome; overlap syndrome;panniculitis; paroxysmal cold hemoglobinuria; paroxysmal nocturnalhemoglobinuria; pemphigoid; pemphigus; pemphigus erythematosus;pemphigus foliaceus; pemphigus vulgaris; pigeon breeder's disease;pneumonitis, hypersensitivity; polyarteritis nodosa; polymyalgiarheumatica; polymyositis; polyneuritis, idiopathic; Portuguese familialpolyneuropathies; pre-eclampsia/eclampsia; primary biliary cirrhosis;progressive systemic sclerosis (scleroderma); psoriasis; psoriaticarthritis; pulmonary alveolar proteinosis; pulmonary fibrosis, Raynaud'sphenomenon/syndrome; Reidel's thyroiditis; Reiter's syndrome, relapsingpolychrondritis; rheumatic fever; rheumatoid arthritis; sarcoidosis;scleritis; sclerosing cholangitis; serum sickness; Sezary syndrome;Sjogren's syndrome; Stevens-Johnson syndrome; Still's disease; subacutesclerosing panencephalitis; sympathetic ophthalmia; systemic lupuserythematosus; transplant rejection; ulcerative colitis;undifferentiated connective tissue disease; urticaria, chronic;urticaria, cold; uveitis; vitiligo; Weber-Christian disease; Wegener'sgranulomatosis; and Wiskott-Aldrich syndrome.

In another aspect, the invention provides a method of enhancing cellularengraftment in a subject comprising administering to a subject in needthereof of a therapeutically effective amount of one or more compoundsof the invention.

As used herein the phrase “enhancing cellular engraftment” refers to animprovement in efficiency, quality or rapidity of cell transplantationwhich may result from improved homing to the target tissue, improvedadhesion, reduced rejection and the like. Methods for assessing cellengraftment potential include, for example, cell migration and other invitro techniques, and histological, immunological and/or radiologicalassessment of tissues and organs from actual in-vivo transplantation. Incertain aspects, the term “enhancing cellular engraftment” alsoincludes, but is not limited to, the treatment of a disorder in whichthe subject is treated with cells, such as in the context of bone marrowtransplantation, stem cell replacement, and skin engraftment.

In another aspect, the invention provides a method of enhancing woundrepair in a subject comprising administering to a subject in needthereof of a therapeutically effective amount of one or more compoundsof the invention.

As used herein the term, “enhancing wound repair” includes, but is notlimited to, the formation of granulation tissue; of wound contraction;and/or the induction of epithelialization (i.e., the generation of newcells in the epithelium). Wound repair is conveniently measured bydecreasing wound area. In certain aspects, the term “enhancing woundrepair” also includes, but is not limited to, the treatment a disorderin which the subject is being treated for tissue damage, either internalor external, such that the goal is to restore organ or tissue integrity.

In another aspect, the invention provides a method of treating aninflammatory condition in a subject comprising administering to asubject in need thereof an effective amount of a composition comprisinga compound of the invention or a pharmaceutically acceptable saltthereof.

As used herein, the term “inflammatory condition” refers to a conditionor disorder associated with one or more aberrant physiological processesor other physiological responses (such as responses to an injurious ornoxious stimulus) which result in a pathophysiological state ofinflammation. An inflammatory condition may be either an acute orchronic inflammatory condition, which can result from infections ornon-infectious causes. Various infectious causes include meningitis,encephalitis, uveitis, colitis, tuberculosis, dermatitis, and adultrespiratory distress syndrome. Non-infectious causes include trauma(burns, cuts, contusions, crush injuries), autoimmune diseases, andorgan rejection episodes. Thus, in specific aspects, an inflammatorycondition results from a condition selected from the group thatincludes: atherosclerosis (arteriosclerosis); autoimmune conditions,such as multiple sclerosis, systemic lupus erythematosus, polymyalgiarheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis,bursitis, psoriasis, fibrosis, arthrosteitis, rheumatoid arthritis andother forms of inflammatory arthritis, Sjogren's Syndrome, progressivesystemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis,dermatomyositis, pemphigus, pemphigoid, Type I diabetes mellitus,myasthenia gravis, Hashimoto's thyroditis, Graves' disease,Goodpasture's disease, mixed connective tissue disease, sclerosingcholangitis, inflammatory bowel disease including Crohn's Disease(regional enteritis) and ulcerative colitis, pernicious anemia,inflammatory dermatoses; usual interstitial pneumonitis (VIP),asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, all thin'sof pneumoconiosis, sarcoidosis (in the lung and in any other organ),desquamative interstitial pneumonia, lymphoid interstitial pneumonia,giant cell interstitial pneumonia, cellular interstitial pneumonia,extrinsic allergic alveolitis, Wegener's granulomatosis and relatedforms of angiitis (temporal arteritis and polyarteritis nodosa); sepsis;inflammatory dermatoses not presumed to be autoimmune; chronic activehepatitis; delayed-type hypersensitivity reactions (e.g., poison ivydermatitis); pneumonia or other respiratory tract inflammation due toany cause; Adult Respiratory Distress Syndrome (ARDS) from any etiology;encephalitis with inflammatory edema; immediate hypersensitivityreactions including, but not limited to, asthma, hayfever, cutaneousallergies, acute anaphylaxis; diseases involving acute deposition ofimmune complexes, including, but not limited to, rheumatic fever, acuteand/or chronic glomerulonephritis due to any etiology, includingspecifically post-infectious (e.g., post-Streptococcal)glomerulonephritis, acute exacerbations of Systemic Lupus Erythematosus;pyelonephritis; cellulitis; cystitis; acute and/or chroniccholecystitis; and conditions producing transient ischemia anywherealong the gastrointestinal tract, bladder, heart, or other organ,especially those prone to rupture; sequelae of organ transplantation ortissue allograft, including allograft rejection in the acute time periodfollowing allogeneic organ or tissue transplantation and chronichost-versus-graft rejection. The term “inflammatory condition” alsoincludes appendicitis, arteritis, blepharitis, bronchiolitis,bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis,dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis,enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis,gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis,myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis,osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis,pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis,salpingitis, sinusitis, stomatitis, synovitis, tonsillitis, uveitis,vaginitis, vasculitis, vulvitis, and vulvovaginitis, angitis, chronicbronchitis, osteomylitis, optic neuritis, temporal arteritis, transversemyelitis, necrotizing fascilitis, hepatitis, and necrotizingenterocolitis.

In a specific aspect, the invention encompasses a method of targetingMCL-1 in a cell comprising contacting as cell with one or more compoundsselected from gossypol, celastrol, manoalide, u73122, bithionol andhexachlorophene. Such targeting may useful in the modulation ofprogrammed cell death or apoptosis, autophagic cell death; necrotic celldeath; cell metabolism; cell division; cell differentiation; RNAtranscription and processing; or protein multimerization ordissociation.

As used herein, the term “targeting” refers to the direct interaction ofthe compound with MCL-1, or to the interaction of the compound with oneor more other factors that in turn interact with MCL-1 or affect MCL-1activity.

Administration

In certain embodiments, a compound of the invention or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered to the subject in a pharmaceutically-acceptableformulation. In certain embodiments, a compound of the invention or apharmaceutically acceptable salt, solvate or hydrate thereof or an acompound of the invention pharmaceutical composition is suitable fortopical, intravenous, parental, or oral administration. The methods ofthe invention further include administering to a subject atherapeutically effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate or hydrate thereof.

The phrase “pharmaceutically acceptable” refers to a compound of theinvention, compositions containing a compound of the invention, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The phrase “pharmaceutically-acceptable carrier” includespharmaceutically-acceptable material, composition or vehicle, involvedin carrying or transporting the subject chemical from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.

Methods of preparing these compositions include the step of bringinginto association a compound of the invention or a pharmaceuticallyacceptable salt, solvate or hydrate thereof with the carrier and,optionally, one or more accessory ingredients. These compositions mayalso contain adjuvants such as preservatives, wetting agents,emulsifying agents and dispersing agents.

Regardless of the route of administration selected, a compound of theinvention, which may be used in a suitable salt, solvate, or hydrateform, and/or the pharmaceutical compositions of a compound of theinvention, are formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art.

Formulations are provided to a subject in an effective amount. The term“effective amount” includes an amount effective, at dosages and forperiods of time necessary, to achieve the desired result. An effectiveamount of a compound of the invention may vary according to factors suchas the disease state, age, and weight of the subject, and the ability ofthe compound to elicit a desired response in the subject. Dosageregimens may be adjusted to provide the optimum therapeutic response.

The effective amount is generally determined by the physician on acase-by-case basis and is within the skill of one in the art. As a rule,the dosage for in vivo therapeutics or diagnostics will vary. Severalfactors are typically taken into account when determining an appropriatedosage. These factors include age, sex and weight of the patient, thecondition being treated, and the severity of the condition. In addition,an effective amount is preferably selected to minimize adverse sideeffects. For example, an effective amount of a compound of the inventionis preferably selected to treat or prevent a disorder as disclosedherein, while minimizing side effects such as hypotension.

Suitable dosages and formulations of a compound of the invention or apharmaceutically acceptable salt, solvate or hydrate thereof can beempirically determined by the administering physician. Standard texts,such as Remington: The Science and Practice of Pharmacy, 17th edition,Mack Publishing Company, and the Physician's Desk Reference, each ofwhich are incorporated herein by reference, can be consulted to preparesuitable compositions and doses for administration. A determination ofthe appropriate dosage is within the skill of one in the art given theparameters for use described herein.

Standard texts, such as Remington: The Science and Practice of Pharmacy,17th edition, Mack Publishing Company, incorporated herein by reference,can be consulted to prepare suitable compositions and formulations foradministration, without undue experimentation. Suitable dosages can alsobe based upon the text and documents cited herein. A determination ofthe appropriate dosages is within the skill of one in the art given theparameters herein.

In terms of treatment, an effective amount is an amount that issufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of an inflammatory condition or other disorder as disclosedherein, or the symptoms thereof. A therapeutically effective amount canbe provided in one or a series of administrations. In terms of anadjuvant, an effective amount is one sufficient to enhance theanti-inflammatory or immune response to the anti-inflammatory orimmunogen. The effective amount is generally determined by the physicianon a case-by-case basis and is within the skill of one in the art.

As a rule, the dosage for in vivo therapeutics or diagnostics will vary.Several factors are typically taken into account when determining anappropriate dosage. These factors include age, sex and weight of thepatient, the condition being treated, the severity of the condition andthe method of administration.

The dosage of a compound of the invention or a pharmaceuticallyacceptable salt, solvate or hydrate thereof can vary from about 0.01 mgto about 1,000 mg per day; about 0.1 mg to about 250 mg per day; about0.5 mg to about 100 mg per day; or about 1 to about 5 mg per day.Ascertaining dosage ranges is well within the skill of one in the art.The dosage of a compound of the invention or a pharmaceuticallyacceptable salt, solvate or hydrate thereof can range from about 0.001to 25 mg/kg of body weight. Such dosages may vary, for example,depending on whether multiple administrations are given, tissue type androute of administration, the condition of the individual, the desiredobjective and other factors known to those of skill in the art.Administrations can be conducted infrequently, or on a regular weeklybasis until a desired, measurable parameter is detected, such asdiminution of disease symptoms. Administration can then be diminished,such as to a biweekly or monthly basis, as appropriate.

A therapeutically effective amount can be administered in one or moredoses. The term “administration” or “administering” includes routes ofintroducing the compound(s) to a subject to perform their intendedfunction. Examples of routes of administration which can be used includeinjection (subcutaneous, intravenous, parenterally, intraperitoneally,intrathecal), oral, inhalation, rectal and transdermal.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound(s), drug or other material,such that it enters the patient's system and, thus, is subject tometabolism and other like processes, for example, subcutaneousadministration.

Such dosages may vary, for example, depending on whether multipleadministrations are given, tissue type and route of administration, thecondition of the individual, the desired objective and other factorsknown to those of skill in the art.

Available routes of administration include subcutaneous, intramuscular,intraperitoneal, intradermal, oral, intranasal, intrapulmonary (i.e., byaerosol), intravenously, intramuscularly, subcutaneously, intracavity,intrathecally or transdermally, alone or in combination with otherpharmaceutical agents.

A cell can be contacted with a composition including a compound of theinvention, in certain embodiments in vivo or, in certain embodiments, invitro. Contacting a cell in vivo can include administration of thecomposition to a subject, or to a tissue, such that a cell is contactedwith a compound of the invention. Contacting a cell in vitro caninclude, e.g., contacting the cell with a compound of the invention, ora composition consisting essentially of a compound of the invention,directly or by addition of irinadalone or the composition to a growthmedium for the cell.

Additional/Secondary Therapeutic Agents

The compounds and compositions of the invention can be administered withone or more additional therapeutic agents either separately or in thesame formulation as a compound of the invention.

In certain embodiments the compounds and compositions of the inventionare useful in sensitizing a cell or a subject to one or more secondarytherapeutic agents. In such embodiments, the invention provides a methodfor sensitizing a cell to a secondary therapeutic agent comprisingadministering to said cell a sensitizing amount of one or more compoundof the invention. A sensitizing amount may be any amount capable ofgenerating a greater response by the cell to the secondary therapeuticagent as compared to the response by the cell without a compound orcomposition of the invention. In certain embodiments a sensitizingamount is a therapeutically effective amount. In other embodiments, asensitizing amount is less than a therapeutically effective amount.

The compounds and compositions of the invention can be administered withone or more additional therapeutic agents either separately or in thesame formulation as a compound of the invention.

Additional Therapeutic agents include, but are not limited to antacids,antibiotics, antiemetic agents, antidepressants, and antifungal agents,anti-inflammatory agents, antiviral agents, anticancer agents,immunomodulatory agents, beta-interferons, hormones or cytokines.

Additional Therapeutic agents further include, but are not limited todeath receptor stimulants (eg. TRAIL), HDAC inhitor (eg. SAHA),proteasome inhibitor (eg. Bortezomib), BCL-2 family inhibitor (eg.ABT-737, ABT-263, obatoclax), kinase inhibitor (e.g. Gleevac, Rafinhibitor), mitotic inhibitors (e.g. Taxol), nucleoside analogs (e.g.Gemcitabine), and anti-cancer monoclonal antibodies (e.g. cetuximab),corticosteroids (e.g. dexamethasone), DNA-damaging agents (e.g.cisplatin), antimetabolites (e.g. methotrexate).

The compounds of the invention can be formulated in combination withantacids. For example, they can be formulated with aluminum carbonate,aluminum hydroxide, bismuth subsalicylate, calcium carbonate, calciumhydroxide, calcium phosphate, dihydroxyaluminum sodium carbonate,magnesium hydroxide, magnesium oxide, magnesium trisilicate, sodiumbicarbonate, simethicone, glycine, or combinations thereof.

The compounds of the invention can be foil mlated in combination withantibiotics. For example, they can be formulated with a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin (e.g.,clarithromycin), an erythromycin (e.g., erythromycin), a penicillin(e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin ornorfloxacin), aminoglycoside antibiotics (e.g., apramycin, arbekacin,bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate,netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin),amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol,florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamideand rifampin), carbacephems (e.g., loracarbef), carbapenems (e.g.,biapenem and imipenem), cephalosporins (e.g., cefaclor, cefadroxil,cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole,cefpiramide, and cefpirome), cephamycins (e.g., cefbuperazone,cefmetazole, and cefminox), monobactams (e.g., aztreonam, carumonam, andtigemonam), oxacephems (e.g., flomoxef, and moxalactam), penicillins(e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,penicillin o-benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin, clinafloxacin,flumequine, and grepagloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

The compounds of the invention can be formulated in combination with anantiemetic agent. Suitable antiemetic agents include, but are notlimited to, metoclopromide, domperidone, prochlorperazine, promethazine,chlorpromazine, trimethobenzamide, ondansetron, granisetron,hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron,benzquinamide, bietanautine, bromopride, buclizine, clebopride,cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine,methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine,thioproperazine, tropisetron, and mixtures thereof.

The compounds of the invention can be formulated or formulated incombination with an antidepressant. Suitable antidepressants include,but are not limited to, binedaline, caroxazone, citalopram, dimethazan,fencamine, indalpine, indeloxazine hydrocholoride, nefopam, nomifensine,oxitriptan, oxypertine, paroxetine, sertraline, thiazesim, trazodone,benmoxine, iproclozide, iproniazid, isocarboxazid, nialamide, octamoxin,phenelzine, cotinine, rolicyprine, rolipram, maprotiline, metralindole,mianserin, mirtazepine, adinazolam, amitriptyline, amitriptylinoxide,amoxapine, butriptyline, clomipramine, demexiptiline, desipramine,dibenzepin, dimetacrine, dothiepin, doxepin, fluacizine, imipramine,imipramine N-oxide, iprindole, lofepramine, melitracen, metapramine,nortriptyline, noxiptilin, opipramol, pizotyline, propizepine,protriptyline, quinupramine, tianeptine, trimipramine, adrafinil,benactyzine, bupropion, butacetin, dioxadrol, duloxetine, etoperidone,febarbamate, femoxetine, fenpentadiol, fluoxetine, fluvoxamine,hematoporphyrin, hypericin, levophacetoperane, medifoxamine,milnacipran, minaprine, moclobemide, nefazodone, oxaflozane, piberaline,prolintane, pyrisuccideanol, ritanserin, roxindole, rubidium chloride,sulpiride, tandospirone, thozalinone, tofenacin, toloxatone,tranylcypromine, L-tryptophan, venlafaxine, viloxazine, and zimeldine.

The compounds of the invention can be formulated in combination with anantifungal agent. Suitable antifungal agents include but are not limitedto amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal,flucytosine, miconazole, butoconazole, clotrimazole, nystatin,terconazole, tioconazole, ciclopirox, econazole, haloprogrin, naftifine,terbinafine, undecylenate, and griseofuldin.

The compounds of the invention can be formulated in combination with ananti-inflammatory agent. Useful anti-inflammatory agents include, butare not limited to, non-steroidal anti-inflammatory drugs such assalicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal,salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin,sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin,ketorolac, dichlofenac, ibuprofen, naproxen, naproxen sodium,fenoprofen, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam,ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone,oxyphenbutazone, antipyrine, aminopyrine, apazone and nimesulide;leukotriene antagonists including, but not limited to, zileuton,aurothioglucose, gold sodium thiomalate and auranofin; steroidsincluding, but not limited to, alclometasone diproprionate, amcinonide,beclomethasone dipropionate, betametasone, betamethasone benzoate,betamethasone diproprionate, betamethasone sodium phosphate,betamethasone valerate, clobetasol proprionate, clocortolone pivalate,hydrocortisone, hydrocortisone derivatives, desonide, desoximatasone,dexamethasone, flunisolide, flucoxinolide, flurandrenolide, halcinocide,medrysone, methylprednisolone, methprednisolone acetate,methylprednisolone sodium succinate, mometasone furoate, paramethasoneacetate, prednisolone, prednisolone acetate, prednisolone sodiumphosphate, prednisolone tebuatate, prednisone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, and triamcinolonehexacetonide; and other anti-inflammatory agents including, but notlimited to, methotrexate, colchicine, allopurinol, probenecid,sulfinpyrazone and benzbromarone.

The compounds of the invention can be formulated in combination withanother antiviral agent. Useful antiviral agents include, but are notlimited to, protease inhibitors, nucleoside reverse transcriptaseinhibitors, non-nucleoside reverse transcriptase inhibitors andnucleoside analogs. The antiviral agents include but are not limited tozidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine,trifluridine, and ribavirin, as well as foscarnet, amantadine,rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir,alpha-interferons; adefovir, clevadine, entecavir, pleconaril.

The compounds of the invention can be formulated in combination with animmunomodulatory agent. Immunomodulatory agents include, but are notlimited to, methothrexate, leflunomide, cyclophosphamide, cyclosporineA, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators, peptidemimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal,polyclonal, Fvs, ScFvs, Fab or F(ab)₂ fragments or epitope bindingfragments), nucleic acid molecules (e.g., antisense nucleic acidmolecules and triple helices), small molecules, organic compounds, andinorganic compounds. Examples of T cell receptor modulators include, butare not limited to, anti-T cell receptor antibodies (e.g., anti-CD4antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® IDEC and SKB), mAB4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies(e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), orRituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linkedimmunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)),anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g.,IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAM:PATH 1H (Ilex)),anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelim (Genentech)),and anti-B7 antibodies (e.g., IDEC-114 (IDEC)) and CTLA4-immunoglobulin.Examples of cytokine receptor modulators include, but are not limitedto, soluble cytokine receptors (e.g., the extracellular domain of aTNF-alpha. receptor or a fragment thereof, the extracellular domain ofan IL-1.beta. receptor or a fragment thereof, and the extracellulardomain of an IL-6 receptor or a fragment thereof), cytokines orfragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-alpha., interferon(WM-alpha., IFN-beta., IFN-gamma, and GM-CSF), anti-cytokine receptorantibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptorantibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptorantibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptorantibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-alpha. antibodies,anti-IL-1beta antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies(e.g., ABX-IL-8 (Abgenix)), and anti-IL-12 antibodies).

The compounds of the invention can be formulated in combination withcytokines. Examples of cytokines include, but are not limited to,interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 interleukin-9(IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin 15(IL-15), interleukin 18 (IL-18), platelet derived growth factor (PDGF),erythropoietin (Epo), epidermal growth factor (EGF), fibroblast growthfactor (FGF), granulocyte macrophage stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), macrophage colonystimulating factor (M-CSF), prolactin, and interferon (IFN), e.g.,IFN-alpha, and IEN-gamma).

The compounds of the invention can be formulated in combination withhormones. Examples of hormones include, but are not limited to,luteinizing hormone releasing hormone (LHRH), growth hormone (GH),growth hormone releasing hormone, ACTH, somatostatin, somatotropin,somatomedin, parathyroid hormone, hypothalamic releasing factors,insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, lowmolecular weight heparins, heparinoids, synthetic and natural opioids,insulin thyroid stimulating hormones, and endorphins.

The compounds of the invention can be formulated in combination withbeta-interferons which include, but are not limited to, interferonbeta-1a and interferon beta-1b.

The compounds of the invention can be formulated in combination with anabsorption enhancer, particularly those which target the lymphaticsystem, including, but not limited to sodium glycocholate; sodiumcaprate; N-lauryl-D-maltopyranoside; EDTA; mixed micelle; and thosereported in Muranishi Crit. Rev. Ther. Drug Carrier Syst., 7-1-33, whichis hereby incorporated by reference in its entirety. Other knownabsorption enhancers can also be used. Thus, the invention alsoencompasses a pharmaceutical composition comprising one or more sulfatedpolysaccharides of the invention and one or more absorption enhancers.

The additional therapeutic agent can act additively or, more preferably,synergistically. In a preferred embodiment, a composition comprising acompound of the invention is administered concurrently with theadministration of another therapeutic agent, which can be part of thesame composition or in a different composition from that comprising thecompounds of the invention. In another embodiment, a compound of theinvention is administered prior to or subsequent to administration ofanother therapeutic agent. In a separate embodiment, a compound of theinvention is administered to a patient who has not previously undergoneor is not currently undergoing treatment with another therapeutic agent,particularly an antiviral agent.

Oral Dosage Forms

A compound of the invention or a pharmaceutically acceptable salt,solvate or hydrate thereof and compositions comprising a compound of theinvention that are suitable for oral administration can be presented asdiscrete dosage forms, such as, but are not limited to, tablets (e.g.,chewable tablets), caplets, capsules, and liquids (e.g., flavoredsyrups). Such dosage forms contain predetermined amounts of activeingredients, and may be prepared by methods of pharmacy well known tothose skilled in the art. See generally, Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an intimate admixture with at least oneexcipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage font's (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing faun such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Aspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,specifically from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, pre-gelatinized starch, otherstarches, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W. R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

Parenteral and Intravascular Dosage Forms

Parenteral and intravascular dosage forms can be administered topatients by various routes including, but not limited to, subcutaneous,intravenous (including bolus injection and constant infusion),intramuscular, and intraarterial. Because their administration typicallybypasses patients' natural defenses against contaminants, parenteral andintravascular dosage forms are preferably sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products (including, but not limited to lyophilizedpowders, pellets, and tablets) ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention.

For intravascular administration, for instance by direct injection intothe blood vessel, or surrounding area, it may be desirable to administerthe compositions locally to the area in need of treatment. This can beachieved, for example, by local infusion during surgery, by injection,by means of a catheter, or by means of an implant, said implant being ofa porous, non-porous, or gelatinous material, including membranes, suchas silastic membranes, or fibers. A suitable such membrane is Gliadel®provided by Guilford Pharmaceuticals Inc.

Transdermal, Topical, And Mucosal Dosage Forms

Transdermal, topical, and mucosal dosage forms of the invention include,but are not limited to, ophthalmic solutions, sprays, aerosols, creams,lotions, ointments, gels, solutions, emulsions, suspensions, or otherforms known to one of skill in the art. See, e.g., Remington'sPhamiaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treatingmucosal tissues within the oral cavity can be formulated as mouthwashesor as oral gels. Further, transdermal dosage forms include “reservoirtype” or “matrix type” patches, which can be applied to the skin andworn for a specific period of time to permit the penetration of adesired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed by this invention are well known to those skilled inthe pharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form lotions, tinctures, creams, emulsions, gelsor ointments, which are non-toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredient of the invention. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, preferredmethods and materials are described below. The materials, methods, andexamples are illustrative only and not intended to be limiting. Otherfeatures and advantages of the invention will be apparent from thedetailed description and from the claims.

Kits

This invention therefore encompasses kits which, when used by themedical practitioner, can simplify the identification of subjects andthe administration of appropriate amounts of a compound of the inventionor a pharmaceutically acceptable salt, solvate or hydrate thereof to apatient.

A typical kit of the invention comprises one or more unit dosage formsof a compound of the invention or a pharmaceutically acceptable salt,solvate or hydrate thereof, and instructions for identification of asubject.

Kits of the invention can further comprise devices that are used toadminister a compound of the invention or a pharmaceutically acceptablesalt, solvate or hydrate thereof. Examples of such devices include, butare not limited to, intravenous cannulation devices, syringes, dripbags, patches, topical gels, pumps, containers that provide protectionfrom photodegradation, autoinjectors, and inhalers.

Kits of the invention can further comprise pharmaceutically acceptablevehicles that can be used to administer one or more active ingredients.For example, if an active ingredient is provided in a solid form thatmust be reconstituted for parenteral administration, the kit cancomprise a sealed container of a suitable vehicle in which the activeingredient can be dissolved to form a particulate-free sterile solutionthat is suitable for parenteral administration. Examples ofpharmaceutically acceptable vehicles include, but are not limited to:Water for Injection USP; aqueous vehicles such as, but not limited to,Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

All documents mentioned herein are incorporated herein by reference intheir entirety.

Examples

In order that the invention may be more fully understood, the followingexamples are provided. It should be understood that these examples arefor illustrative purposes only and are not to be construed as limitingthe invention in any way.

General Experimental Methodology

-   -   1. To identify potent and selective small molecule modulators of        MCL-1, a small molecule binding screen by competitive        fluorescence polarization assay was conducted. Small molecules        were screened in parallel against the FITC-MCL-1 SAHB/MCL-1ΔNΔC        interaction and the FITC-BAD BH3/BCL-X_(L)AC interaction. Those        compounds that preferentially disrupted the FITC-MCL-1        SAHB/MCL-1ΔNΔC interaction were advanced to secondary screening        analyses.    -   2. A series of confirmatory binding assays were performed.        First, the competitive binding assay using FITC-MCL-1        SAHB/MCL-1ΔNΔC was repeated using dose-response analysis to        confirm dose-responsive binding and determine small molecule        K_(i) values. Subsequently specificity analysis was performed by        competitive fluorescence polarization binding assay of small        molecules against the interactions of FITC-BID BH3 in        combination with MCL-1ΔNΔC, BCL-2ΔC, BCL-X_(L)ΔC, BCL-wΔC, and        BFL1/A1ΔC. Small molecules exhibiting high fidelity specificity        for MCL-1ΔNΔC over the other anti-apoptotic proteins were        advanced to SAR, binding site, and functional analysis. Docking        analysis was performed to confirm the compatibility of small        molecule structure with an energetically favorable docking site        at the structurally defined (i.e. x-ray crystallography)        BH3-binding pocket of MCL-1ΔNΔC. Dilutional assays were        performed to distinguish between non-covalent and covalent MCL-1        binders. These studies identified a series of covalent        interactors; for example, a novel site of interaction for        inhibiting MCL-1 binding activity was identified based on        covalent modification of MCL-1 by the molecule at cysteine 286.        Finally, selective MCL-1 inhibitors were tested in a        BAX-mediated liposomal release assay to evaluate their capacity        to block the inhibitory/anti-apoptotic activity of MCL-1.    -   3. Small molecule modulators of MCL-1 were then tested in a        cellular apoptosis sensitization assay using OPM2 cells exposed        to subcytotoxic doses of TRAIL in the presence or absence of        serial dilutions of small molecule. Sensitization activity was        also confirmed by use of CalcuSyn analysis of combination        treatments.

Methods SAHB Synthesis

Hydrocarbon-stapled peptides corresponding to BCL-2 family BH3 domainsand their FITC-βAla derivatives were synthesized, purified, andcharacterized using previously described methodologies^(24,27,29).

Anti-Apoptotic Protein Preparation

Recombinant and tagless MCL-1ΔNΔC, BCL-2 ΔC, BCL-XLΔC, BCL-wΔC, andBFL1/A1ΔC were produced using previously described methods^(27,29,31).

Small Molecule Screening

Small molecule screens were performed at the Institute for Chemistry andCellular Biology at Harvard Medical School and employed commerciallibraries (Asinex, Chembridge, ChemDiv, Enamine, Life Chemicals, andMaybridge). A high-throughput competitive FP binding assay was employedto screen for small molecules that disrupted the interaction betweenFITC-MCL-1 SAHB (15 nM) and MCL-1ΔNΔC (45 nM). MCL-1ΔNΔC was expressedand purified by FPLC and delivered by automated liquid handler to 384well plates, followed by addition of small molecule libraries. After a15 minute incubation at room temperature, FITC-MCL-1 SAHB (15 nM) wasadded to each well by liquid handler and FP read at equilibrium (e.g. 1hr). Small molecule hits were re-examined in this assay using serialdilutions of the compounds to confirm dose-responsive inhibition ofFITC-MCL-1 SAHB binding.

Confirmatory Binding Analyses

The most potent hits were advanced to rigorous quantification of bindingactivity and specificity. First, serial dilutions of small molecule intriplicate were mixed with FITC-MCL-1 SAHB (15 nM), followed by additionto 384-well black Costar plates containing MCL-1ΔNΔC (45 nM) diluted inbinding buffer (50 mM Tris, 100 mM NaCl, 0.0625% CHAPS, pH 8.0). Theplates were incubated in the dark at room temp until equilibrium wasreached (i.e. stabilization of binding isotherms) and FP (mP units)determined using a microplate reader (e.g. Spectramax). K_(i) valueswere calculated by nonlinear regression analysis of dose-response curvesusing Prism software (Graphpad). Small molecule hits were then testedfor their ability to dissociate FITC-BAK BH3 from MCL-1ΔNΔC in acompetitive FP assay, performed as described above except that smallmolecule dilutions were added to a solution of FITC-BAK BH3 (25 nM) andMCL-1ΔNΔC (250 nM) to simulate a physiologic displacement ofpro-apoptotic BAK from the inhibitory MCL-1 protein. For rigorousspecificity analysis, the identical competitive FP-type experiment wasperformed except that serial dilutions of small molecule hits were mixedwith the pan-anti-apoptotic binder FITC-BID BH3 (15 nM), followed byaddition to plates containing either MCL-1ΔNΔC, BCL-2ΔC, BCL-X_(L)ΔC,BCL-wΔC, or BFL1/A1ΔC. We used pGEX vectors to express GST-MCL-1ΔNΔC,BCL-2ΔC, BCL-X_(L)ΔC, BCL-wΔC, and BFL1/A1ΔC, followed by thrombincleavage and FPLC-based gel filtration chromatography. Of note, theC-terminal alpha-helix was deleted from each of these constructs tofacilitate protein expression and purification; an additional N-terminaldeletion of MCL-1 was performed to further enhance expression, purity,and stability. FP analysis was performed as above and those moleculesshowing exclusive binding activity for MCL-1ΔNΔC were advanced tostructural docking and functional testing.

Dilution Assay

MCL-1ΔNΔC (2 μM, 500 mL) was incubated with compound (at a ratio wellabove each previously determined EC₅₀ value) for 1 hour at roomtemperature in FP buffer (100 mM NaCl, 50 nM Tris, pH 8). A small samplewas taken as a pre-dilution control. Compound-treated protein andunmodified protein were then diluted to a volume of 20 mL, andsubsequently re-concentrated to 500 mL using 10K centrifugal filterunits (Amicon). FP binding analysis was performed as described above byadding serial dilutions of compound-modified MCL-1 and unmodified MCL-1,both before and after dilution, followed by FITC-BID BH3 (15 nM) to a96-well black flat bottom plate (Costar). FP measurements and analysesare then performed as described above.

Identification of Covalent Modification Site by Mass Spectrometry

MCL-1ΔNΔC (90 μM) was incubated with a 1:1.25 ratio of compound for 2hours at room temperature in 150 mM NaCl, 50 mM Tris, pH 7.4. Excesscompound was removed by gel filtration and the protein was digested withtrypsin overnight at 37° C. Peptides were analyzed by nano-LC/ESI/MSusing a vented column assembly as described (Ficarro et al., 2009)³⁴.Briefly, peptides were injected using an autosampler and HPLC (WatersNanoAcquity) onto a self-packed precolumn (4 cm, 100 μm I.D., POROS10R2,Applied Biosystems) and gradient eluted (0-30% B in 20 minutes, A=0.2 Macetic acid in water, B=acetonitrile with 0.2M acetic acid) to theresolving column (self-packed 30 μm I.D., 12 cm of 5 μm Monitor C18,Column Engineering) and introduced to the mass spectrometer (ThermoFisher LTQ-Orbitrap XL) via ESI (spray voltage=2.2 kV). The top 8 mostabundant precursors in each MS scan (image current detection,resolution=30,000) were subjected to CAD (electron multiplier detection,collision energy=35%). A separate targeted nano-LC/ESI/MS experiment wasperformed with compound-modified peptide (e.g. TINQES*CIEPLAESITDVLVR[*C=modified cysteine)]), which was subjected to HCD (higher energycollisionally activated dissociation).

Liposomal Release Assay

Liposomes are prepared from a mixture of lipids that reflect thecomposition of the outer mitochondrial membrane as previously describedin detail (Pitter et al., 2008)³¹. Aliquots of mixed lipids (1 mg total)are stored in glass at −20° C. under nitrogen, and before use,resuspended in liposome assay buffer (10 mM HEPES, 200 mM KCl, 1 mMMgCl₂, pH 7) with 12.5 mM of the fluorescent dye ANTS(8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt) and 45 mM ofthe quencher DPX (p-xylene-bis-pyridinium bromide). The resulting slurryis vortexed for 10 minutes and freeze-thawed five times alternatingbetween liquid nitrogen and a 40° C. water bath. The solution is thenpassed through an Avanti Mini-Extruder Set (#610000) equipped with a100-nm filter, followed by passage through a Sepharose column (GEHealthcare) to remove residual ANTS/DPX. The lipsomes are brought up toa volume of 3 mL to produce a final liposome stock. For the liposomalrelease assay, a total volume of 30 μL is used in 384 well black flatbottom plates (Costar), and baseline fluorescence measurements of 8 μLliposomes are made for 10 minutes using the Tecan Infinite M1000(excitation: 355 nm, emission: 520 nm). Following the baseline read,recombinant MCL-1ΔNΔC pre-incubated with a 1:1 ratio of compound isadded to the liposomes. Next, 20 nM caspase-cleaved mouse BID (R&Dsystems) and 250 nM purified recombinant monomeric BAX is added, andfluorescence measurements (F) are recorded every minute from time zero(F0) until the release readings plateau. The liposomes are then quenchedwith 1% Triton X-100 (100% release; F100), and percent ANTS/DPX releaseis calculated as ((F−F0)/(F100−F0))×100.

Cytochrome c Release Assays

Mouse liver mitochondria (0.5 mg/mL) are isolated and release assaysperformed as described³¹. Mitochondria are incubated with a serialdilution of MCL-1 targeting small molecule, singly or in combinationwith BID BH3, and after 40 minutes, the pellet and supernatant fractionsare isolated and cytochrome c quantitated using a colorimetric ELISAassay (R&D Systems). Percent cytochrome c released into the supernatant(% cytoc_(sup)) from releasable mitochondrial pools was calculatedaccording to the following equation: %cytoc=[(cytoc_(sup)−cytoc_(backgr))/(cytoc_(total)−cytoc_(backgr))]*100,where background release represents cytochrome c detected in thesupernatant of vehicle-treated (1% DMSO) samples and total releaserepresents cytochrome c measured in 1% Triton-X 100 treated samples. Allexperimental conditions are also tested on Bak^(−/−) mitochondria toensure that the observed cytochrome c release from wild-typemitochondria derives from BAK activation.

Immunoprecipitation Assay

MCL-1-expressing cancer cells (10×10⁶) are incubated with the MCL-1targeting small molecule or vehicle in serum-free media at 37° C. for 4hours, followed by serum replacement for an additional 6 hours. Aftercellular lysis in 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM DTT,0.5% NP40 and complete protease inhibitor pellet, cellular debris ispelleted at 14,000 g for 10 minutes at 4° C. The supernatant is exposedto pre-equilibrated protein A/G sepharose beads and the pre-clearedsupernatant subsequently incubated with anti-MCL-1 antibody for 1.5hours at 4° C., followed by the addition of protein A/G sepharose beadsfor 1 hour. The beads are pelleted and washed with lysis buffer for 10minutes at 4° C. The washed bead are then pelleted, heated to 90° C. for10 minutes in SDS loading buffer, analyzed by SDS/PAGE, and thenimmunoblotted for MCL-1 and BAK.

Crystallography

Crystallization conditions for lead small molecule-MCL-1ΔNΔC complexesare screened using 96-well sitting drop plates set up using a Phoenixcrystallization robot. Initial conditions include HT Index Screen, JSCG+Suite, and Pro-Complex Suite. Screening around the best hit, includingvarying pH, salt, and detergent concentrations, are performed toidentify the best condition for crystal growth. Once generated, thecrystals are removed, washed in the crystallization buffer, andsubjected to mass spectroscopy to verify the presence of compound andprotein within the crystal. The crystal is then soaked incyroprotectant, flash frozen, and stored in liquid nitrogen. Suitablecrystals are examined at the Argonne National Laboratory synchrotronfacility. Phases are obtained by molecular replacement followed by dataanalysis and refinement (Phaser, Phenix, and Coots software).

Cellular Apoptosis Induction Assays

Small molecule inhibitors of MCL-1 were screened in MCL-1-expressingcancer cell lines, such as OPM-2 (multiple myeloma) cells. The cells aretreated with the small molecule MCL-1 inhibitor, alone or in combinationwith subtherapeutic pro-apoptotic stimuli (e.g. TRAIL, Fas ligand,ABT-737), and then cell viability is measured at 48 hours by MTT assayperformed according to the manufacturer's protocol (Roche) andquantitated by an ELISA microplate reader (Biorad). IC₅₀ values weredetermined by nonlinear regression analysis using Prism software(Graphpad). Small molecules that decrease cell viability are thenscreened for cellular apoptosis induction by annexin V binding and FACSanalysis, and by cell fractionation-based mitochondrial cytochrome crelease, as described²⁸. Apoptosis is also correlated with in situdissociation of the MCL-1/BAK complex (immunoprecipitation) performed asdescribed above. As an additional measure of small molecule MCL-1specificity, the identical experiments are conducted on wild-type vs.Mcl-1^(−/−) MEFs, to explicitly link the selective MCL-1 targetingcapacity of the small molecules to sensitization of apoptosis. Thecaspase-dependency of small molecule activity is also confirmed bymonitoring for blockade of activity upon co-treatment with thepan-capase inhibitor Z-VAD.

Pharmacokinetic Analyses

Lead small molecules undergo pharmacokinetic (PK) analysis in mice,performed in conjunction with the DF/HCC Clinical Pharmacology Core.LC/MS-based analytical assays are developed in order to detect andquantify compound levels in plasma. For PK analysis, small molecules(e.g. 10, 50, 100 mg/kg) are injected by tail vein or intraperitoneallyinto male C57/BL6 mice. Blood samples are withdrawn by retro-orbitalbleed at various time points and plasma isolated for compoundquantification, followed by calculation of plasma half-life, peak plasmalevels, total plasma clearance, and apparent volume of distribution.Small molecules that exhibit selective MCL-1 targeting in cells andexhibit a favorable pharmacokinetic profile re advanced to in vivotesting.

In Vivo Efficacy Studies

Small molecule-sensitive cancer cell lines are retrovirally transducedto achieve stable luciferase expression (pMNP-LucNeo) and transplantedinto SCID beige mice as previously described^(27,32). Initial xenograftstudies examine 5 mouse cohorts (n=10), treated with either vehiclealone, low or high dose small molecule alone, or low/high dose smallmolecule in combination with subtherapeutic dosing of pro-apoptoticstimuli (e.g. TRAIL, ABT-737, doxorubicin, etoposide, dexamethasone).Starting on experimental day 1, mice receive a once daily tail injectionof small molecule (e.g. 25 or 100 mg/kg, with or without combinationtreatment). For alternate day in vivo tumor imaging, mice areanesthetized with inhaled isoflurane and treated concomitantly withintraperitoneal injection of D-luciferin. Photonic emission is imaged (2min exposure) using a Xenogen In Vivo Imaging System and total bodybioluminescence quantified by the integration of photonic flux(photons/sec) using Xenogen's Living Image Software. The survivaldistributions of experimental mice are determined using the Kaplan-Meiermethod and compared using the log-rank test. The Fisher's exact test isused to compare the proportion of mice who fail treatment, wheretreatment failure is defined as progression or death, and success asstable disease or regression. If a treatment response is observed with aparticular small molecule, three additional cohorts, treated with eithervehicle, small molecule, or small molecule combination will be used forpharmacodynamic studies in which pro-apoptotic activity is evaluated intissues by TUNEL and activated caspase-3 immunohistochemical staining.

EXAMPLES Identification of MCL-1 Selective Small Molecules by aCompetitive Fluorescence Polarization Binding Assay Using FITC-MCL-1SAHB and MCL-1ΔNΔC.

A high-throughput competitive FP binding assay was employed to screenfor small molecules that disrupted the interactions between FITC-MCL-1SAHB/MCL-1ΔNΔC and FITC-BAD BH3/BCL-X_(L)AC (FIG. 1). Compounds wereranked based upon their selectivity for MCL-1ΔNΔC over BCL-X_(L)ACtargeting, and tabulated (see Table 1 (including Subtables Table 1-Athrough Table 1-P)).

The Structures of Small Molecule/MCL-1 Complexes Provide a Template forRefining Mcl-1 Binding Activity and Specificity.

Using the crystal structure of MCL-1ΔNΔC, the identified small moleculeswere docked onto the BH3-binding pocket of MCL-1ΔNΔC to analyze thelocation and energetic favorability of interaction. The compoundscovered discrete subregions of BH3-binding pocket topography (FIG. 2),providing a blueprint for engineering selective, larger molecules thatincorporate combinations of molecules or their subfragments to engagethe complex and extended BH3-binding surface of MCL-1.

Subclassification of Selective MCL-1 Inhibitors Based on Non-Covalentvs. Covalent Interactions.

To examine the mechanism of small molecule binding to MCL-1, thecompounds were subjected to a rapid dilution assay (FIG. 2C). Briefly,MCL-1ΔNΔC was pre-incubated with the small molecule inhibitors prior todilution with buffer. The solution was concentrated to its originalvolume and fluorescence polarization was performed. Compounds that bindcovalently will remain bound to the protein, whereas compounds that bindreversibly will be exchanged upon dilution. As a positive control, therapid dilution assay confirmed that gossypol (1570G15) bound reversiblyto MCL-1ΔNΔC (FIG. 2C, top panel). In contrast, 1725P16 and 1597E07failed to exchange upon dilution, indicating that the compounds bind andcovalently modified MCL-1ΔNΔC (FIG. 2C, middle, bottom panels).

Identification of a Novel Interaction Site for MCL-1 Inhibition Based onC286 Engagement.

To localize sites of covalent modification, small molecule-treatedMCL-1ΔNΔC (e.g. 1929A19) was subjected to mass spectrometry. Anadditional MALDI peak was observed for small molecule-treated protein,with the increase in mass corresponding to the molecular weight of thecovalently tethered compound. The MCL-1ΔNΔC-adduct was further subjectedto tandem MS/MS, which revealed cysteine 286 as the site of modificationfor 1929A19 (FIG. 2D). Importantly, cysteine 286 is not located at thecanonical BH3 binding pocket (FIG. 2D), indicating that engagement ofthis novel binding site can allosterically regulate MCL-1'santi-apoptotic/BH3-binding activity. To confirm the functionalimportance of C286 engagement, a C286S construct of MCL-1ΔNΔC wasgenerated by site-direct mutagenesis and small molecule binding activitywas compared in competitive FP assays using FITC-BID BH3 and thewild-type and mutant MCL-1ΔNΔC proteins. Whereas 1929A19 and 1616N08competed with FITC-BID BH3 for MCL-1ΔNΔC binding, the molecules failedto engage the C286S construct of MCL-1ΔNΔC, as demonstrated by thecapacity of FITC-BID BH3 to maintain binding activity toward MCL-1ΔNΔCC286S in the presence of the compounds.

Confirmation of Small Molecule Binding Activity and Selectivity by Useof Competitive Binding Assays.

To verify the capacity of the identified small molecules to selectivelytarget MCL-1ΔNΔC, small molecule hits were tested for their differentialability to dissociate FITC-BID BH3 from a panel of anti-apoptoticproteins, including MCL-1ΔNΔC C, BCL-2ΔC, BCL-XLΔC, BCL-wΔC, andBFL1/A1ΔC. As exemplified in FIGS. 3 and 4, small molecules identifiedas MCL-1ΔNΔC selective indeed exhibited preferential displacement ofFITC-BID BH3 from MCL-1ΔNΔC compared to the other anti-apoptoticproteins tested.

Structure-Activity Relationship Analysis of Selective MCL-1 Inhibitors.

Chemical derivatives of class A (FIG. 4C) and J (FIG. 4D) moleculesdemonstrated significant differences in binding activity. Importantly,the five-member thiotriazole ring at position R1 of class A increasedbinding affinity for MCL-1ΔNΔC compared to smaller, non-aromaticsubstituents. At the R2 position, aromatic residues were favored withsmall hydrophobic side chains or hydrogen bond acceptors at thepara-position of the phenyl ring. For class J, small aliphatic groupswere well tolerated at the R1 position, and the trihydroxyphenyl ring atthe R2 position demonstrated superior binding affinity for MCL-1ΔNΔC.Finally, five- or six-member aromatic rings were favored at the R3position. Specifically, analogs that contained methyl substituents at R3displayed no activity unless paired with a large aliphatic group at theR1 position.

Small Molecule MCL-1 Binders Sensitize Cancer Cells to Pro-ApoptoticStimuli.

Cancer cells that depend on MCL-1 for survival were treated withselective small molecule MCL-1ΔNΔC binders in combination with otherpro-apoptotic agents to assess for synergistic anti-tumor activity. Asexemplified in FIG. 5, small molecule bioactives synergized with TRAILto kill OPM2 multiple myeloma cells, as assessed by MTT viability assayand by combination treatment analysis using CalcuSyn software.

Selective MCL-1 Inhibitors Block the Anti-Apoptotic Function of MCL-1 ina BAX-Mediated Liposomal Release Assay.

The liposomal release assay is designed to mimic the functional releaseof mitochondrial cytochrome c by formation BAX-containing pores withinthe outer mitochondria membrane. Here, recombinant full length BAX wasincubated with recombinant tBID in the presence or absence of MCL-1ΔNΔC.As demonstrated in FIG. 6, tBID triggered BAX-mediated release of thefluorophore from the liposome in a time-dependent manner. MCL-1ΔNΔCblocked BAX activation, inhibiting fluorophore release. By inhibitingMCL-1ΔNΔC, 1929A19 and 1616N08 dose-responsively re-instatedtBID-induced, BAX-mediated liposomal release of the fluorophore,highlighting the capacity of the small molecule MCL-1 inhibitors toblock the anti-apoptotic activity of MCL-1.

TABLE 1-A MCL-1% BCL-XL % Spec Structure Bound Bound Values

34.13126397 102.3629331 68.231669

16.68871081 105.34160

34.54623163 100.760843 66.214611

35.01128141 106.8345939 71.823312

56.60157162 106.2747212 49.67315

10.32290702 104.1661092 93.843202

1.259544221 90.57802572 89.318482

27.20036677 100.4228001 73.222433

3.955080904 101.0243434 97.069262

41.01271009 100.3606236 59.347914

32.90443509 97.09434568 64.189911

15.93536133 98.2573418 82.32198

indicates data missing or illegible when filed

TABLE 1-B

R1 = hereocycle or aromatic R2 = H or S- heterocycle or aromaticStructure MCL-1% Bound BCL-XL % Bound Spec Values

2.332623997 103.4341651 101.1015411

4.951710591 96.11650485 91.16479426

19.67020024 96.38730675 76.71710652

19.74954984 93.16376474 73.4142149

35.6 98.3 62.7

36.15465782 91.34577213 55.19111431

TABLE 1-C

where R3 = heterocycle or aromatic Structure MCL-1% Bound BCL-XL % BoundSpec Values

9.463295931 115.1848233 105.7215274

−0.3 108.4 108.7

42.48636709 113.7228826 71.23651547

27.16644285 93.0177803 65.85133746

43.7012494 106.4084449 62.70719545

58.33277892 103.7511722 45.41839332

TABLE 1-D

Structure MCL-1% Bound BCL-XL % Bound Spec Values

14.6 101.3 86.7

−2.04714193 75.96629122 78.01343315

8.332108744 81.17127433 72.83916559

43.2 109.5 66.3

35.72624681 99.74101397 64.01476715

37.6 98.3 60.7

45.37762426 98.52069172 53.14306746

TABLE 1-E

where R1 = amide or ester R2 = heterocycle or aromatic Structure MCL-1%Bound BCL-XL % Bound Spec Values

−0.733372059 80.39676939 81.13014145

11.72800505 79.87789988 68.14989482

31.16639478 95.42028511 64.25389033

38.47471452 101.4501065 62.97539199

18.74037573 77.59057823 58.8502025

47.00574838 99.25113873 52.24539035

53.4905597 105.6295695 52.13900976

47.95352324 96.90361633 48.95009309

56.33127545 104.4315992 48.10032378

55.14812663 103.2306124 48.08248577

64.63766028 112.7047864 48.0671261

62.7184466 110.0825659 47.36411929

40.50995892 86.74650699 46.23654806

66.09053804 111.9999168 45.90937874

56.10428305 101.763264 45.65898096

69.53461354 115.1985743 45.66396079

68.79611916 114.1804937 45.38437457

TABLE 1-F

Structure MCL-1% Bound BCL-XL % Bound Spec Values

14.30073607 84.68236855 70.38163248

25.28012412 92.88964225 67.60951814

19.17808219 73.82692962 54.64884742

33.27874504 82.61218664 49.33344159

TABLE 1-G

where R1 = heterocycle or aromatic Structure MCL-1% Bound BCL-XL % BoundSpec Values

24.59486004 99.32600497 74.73114493

18.96382387 92.90700473 73.94318087

15.16614831 84.81906443 69.65291612

30.48780488 79.68386424 49.19605936

TABLE 1-H

where R1 = aromatic or heterocycle R2 = CH2 Structure MCL-1% BoundBCL-XL % Bound Spec Values

−10.5954323 87.16205145 97.75748375

7.037265168 97.35012677 90.3128616

3.36867863 86.24446993 82.8757913

−7.07177814 75.49565787 82.56743601

20.58823529 102.1918705 81.60363516

30.19710502 107.6879038 77.4907988

21.8 98.2 76.4

15.04465661 89.36779259 74.32313598

31.4290114 89.23693465 57.80792326

30.81305821 84.78776478 53.97470658

36.80576864 89.70132202 52.89555338

38.82044965 91.59237753 52.77192788

29.33477056 78.76829966 49.43352911

TABLE 1-I

where R1 = H or sulfonamide-, ester- or amide linked heterocycle oraromatic R2 = H or aromatic Structure MCL-1% Bound BCL-XL % Bound SpecValues

−5.219264716 104.6841563 109.903421

11.19902121 76.15107324 64.95205204

25.38058602 86.7447645 61.36417848

44.3557943 92.23836973 47.88257544

TABLE 1-J

where X = X or O R3 = aromatic or heterocycle Structure MCL-1% BoundBCL-XL % Bound Spec Values

19.08127208 95.08543531 76.00416323

43.51306119 106.9095962 63.39653504

39.32183657 100.3628503 61.04101373

41.8167411 102.1683264 60.35158527

38.17542436 95.91685226 57.74142791

45.19897498 102.0166733 56.81769831

41.985733 98.20470972 56.21897672

52.01193335 105.6743612 53.6624278

46.42363385 99.00409172 52.58045787

39.96352925 92.21643287 52.25290362

30.59011861 81.34023006 50.75011146

33.00999412 82.65729799 49.64730387

51.18862475 99.22340759 48.03478284

55.45434348 100.8550361 45.4006926

TABLE 1-K

where R1 = phenyl or furan X = N or C Structure MCL-1% Bound BCL-XL %Bound Spec Values

2.606828112 96.58770003 93.98087192

15.57226143 89.4401828 73.86792137

48.21516581 111.214653 62.99948715

43.16379248 101.2275149 58.06372239

13.28219273 66.62503346 53.34284073

48.95344507 97.58836088 48.63491581

215.6015433 263.737406 48.13586273

40.42192793 87.79749734 47.37556941

TABLE 1-L

where X1 = O or S X2 = S or N Structure MCL-1% Bound BCL-XL % Bound SpecValues

−9.610778443 87.82745228 97.43823072

19.68883273 102.4094424 82.72060968

33.27029276 112.3457833 79.07549058

26.07784431 102.7683663 76.69052194

19.8 95.8 76.1

38.57217917 107.8998073 69.32762815

21.28742515 90.20742087 68.91999572

23.0 89.9 66.9

29.45492662 92.21556886 62.76064224

27.92957859 85.64814815 57.71856956

38.04746419 93.34466638 55.29720219

21.54074074 76.08572124 54.5449805

42.5929832 94.16497422 51.57199103

39.83535868 91.73339804 51.89803936

48.7076967 98.89474361 50.1870469

58.50862999 105.8574397 47.34880968

TABLE 1-M

where R1 = aromatic or heterocycle Structure MCL-1% Bound BCL-XL % BoundSpec Values

−23.10049446 101.7468716 124.8473661

−16.27483907 89.94614004 106.2209791

TABLE 1-N MCL-1% BCL-XL % Spec Structure Bound Bound Values

−65.83063646 102.6892674 168.5199038

−6.59173964 129.3090909 135.9008305

−23.10049446 101.7468716 124.8473661

−30.96202705 88.78573776 119.7477648

−5.219264716 104.6841563 109.903421

−0.3 108.4 108.7

−1.785938876 106.6729323 108.4588712

−11.55743971 96.56812237 108.255621

−16.27483907 89.94614004 106.2209791

9.463295931 115.1848233 105.7215274

−7.035327937 98.55501099 105.5903389

10.7155014 114.7223418 104.0068404

2.332623997 103.4341651 101.1015411

14.04124001 112.8657315 98.82449146

−10.86859971 87.36357022 98.23216992

−14.50299142 83.61559031 98.11858173

−10.5954323 87.16205145 97.75748375

−9.610778443 87.82745228 97.43823072

3.955080904 101.0243434 97.0692625

−18.5 78.1 96.6

2.606828112 96.58770003 93.98087192

10.32290702 104.1661092 93.84320213

6.6 99.9 93.2

4.951710591 96.11650485 91.16479426

7.037265168 97.35012677 90.3128616

1.259544221 90.57802572 89.3184815

14.82516366 103.9217319 89.09656823

16.68871081 105.3416001 88.65288929

14.6 101.3 86.7

−13.00667806 71.10363607 84.11031413

30.17353579 114.1379598 83.964424

16.19966207 99.90532748 83.70566541

9.240186268 92.49338093 83.25319466

3.36867863 86.24446993 82.8757913

19.68883273 102.4094424 82.72060968

20.72333282 103.4206261 82.69729329

6.516370589 89.09642401 82.58005342

−7.07177814 75.49565787 82.56743601

15.93536133 98.2573418 82.32198048

20.58823529 102.1918705 81.60363516

−0.733372059 80.39676939 81.13014145

22.9770388 104.0060981 81.02905928

11.4 92.2 80.8

−3.913592369 75.92785571 79.84144808

21.93834459 101.5900478 79.65170319

33.27029276 112.3457833 79.07549058

−2.04714193 75.96629122 78.01343315

30.19710502 107.6879038 77.4907988

19.67020024 96.38730675 76.71710652

13.3744856 90.06707122 76.69258563

26.07784431 102.7683663 76.69052194

21.8 98.2 76.4

17.4 93.7 76.4

19.8 95.8 76.1

19.08127208 95.08543531 76.00416323

21.79034158 97.16842962 75.37808804

21.55274889 96.34785598 74.79510709

24.59486004 99.32600497 74.73114493

15.04465661 89.36779259 74.32313598

18.96382387 92.90700473 73.94318087

15.57226143 89.4401828 73.86792137

19.74954984 93.16376474 73.4142149

27.20036677 100.4228001 73.22243335

37.92480367 111.0248078 73.10000413

30.22510381 103.1419548 72.916851

8.332108744 81.17127433 72.83916559

12.58907363 84.89878885 72.30971522

35.01128141 106.8345939 71.8233125

42.48636709 113.7228826 71.23651547

−5.849688118 65.32554791 71.17523603

14.49140445 85.26500639 70.97360194

58.7 129.5 70.8

11.97648788 82.61082938 70.6343415

14.30073607 84.68236855 70.38163248

−7.305502846 62.96737927 70.27288212

15.16614831 84.81906443 69.6529161

38.57217917 107.8998073 69.32762815

38.64682418 107.8504978 69.20367362

21.28742515 90.20742087 68.91999572

31.28187691 100.1843318 68.90245488

19.37221111 87.66541823 68.29320712

14.6 82.8 68.3

34.13126397 102.3629331 68.23166909

11.72800505 79.87789988 68.14989482

39.1 107.0 67.9

25.28012412 92.88964225 67.60951814

34.53258845 102.162884 67.43869999

23.0 89.9 66.9

37.92480367 104.5573433 66.6325396

43.2 109.5 66.3

30.47645315 96.72727273 66.25081958

34.54623163 100.760843 66.2146114

23.45468053 89.61072441 66.15604388

24.12504492 90.1231625 65.99811758

27.16644285 93.0177803 65.85133746

−8.097006083 57.6845011 65.78150718

66.13604597 131.2029473 65.06690133

11.19902121 76.15107324 64.95205204

4.577793748 69.52721494 64.94942119

18.95094399 83.66485345 64.71390946

26.7 91.4 64.7

34.3965106 99.00834642 64.61183582

31.16639478 95.42028511 64.25389033

32.90443509 97.09434568 64.18991059

36.11902501 100.2785294 64.15950436

35.72624681 99.74101397 64.01476715

21.78361457 85.37461774 63.59100317

39.7488359 103.3154122 63.56657629

41.28528975 104.7348973 63.44960756

43.51306119 106.9095962 63.39653504

18.57176712 81.71632703 63.14455991

29.27084059 92.34063996 63.06979937

31.39551493 94.41842001 63.02290508

48.21516581 111.214653 62.99948715

27.71898883 90.70584478 62.98685595

38.47471452 101.4501065 62.97539199

−19.61126884 43.27239348 62.88366232

6.012957705 68.85183202 62.83887431

29.45492662 92.21556886 62.76064224

35.6 98.3 62.7

43.7012494 106.4084449 62.70719545

40.93873164 103.0201067 62.08137505

31.21264846 93.01166131 61.79901285

39.52157023 101.2560945 61.73452431

59.441744 120.9256794 61.48393538

25.38058602 86.7447645 61.36417848

39.32183657 100.3628503 61.04101373

18.43595701 79.39308398 60.95712697

−29.64270278 31.23996145 60.88266423

27.27621091 88.11755764 60.84134673

37.6 98.3 60.7

4.673280876 65.02894576 60.35566489

41.8167411 102.1683264 60.35158527

39.93161377 99.39343581 59.46182205

41.01271009 100.3606236 59.34791354

47.18965126 106.5320264 59.34237511

36.55154869 95.64034877 59.08880009

41.78912826 100.8274721 59.03834381

34.66750489 93.68924174 59.02173685

18.74037573 77.59057823 58.8502025

22.81374655 81.47543659 58.66169004

39.52194519 98.09873249 58.5767873

39.11594696 97.63720151 58.52125455

30.40891164 88.85677858 58.44786694

25.20612485 83.4987795 58.29265464

43.16379248 101.2275149 58.0372239

31.4290114 89.23693465 57.80792326

7.609675481 65.35257732 57.74290184

38.1742436 95.91685226 57.74142791

27.92957859 85.64814815 57.71856956

48.94267421 106.2615101 57.31883592

32.32116549 89.4407446 57.11957911

38.73289121 95.62561095 56.89271974

45.19897498 102.0166733 56.81769831

35.39582849 92.03372469 56.6378962

34.63367974 90.94954311 56.31597337

44.3733406 100.6715454 56.29820482

41.985733 98.20470972 56.21897672

45.61615856 101.6646949 56.04853639

41.13775626 97.06777083 55.93001457

7.821811681 63.5443038 55.72249212

45.56074766 101.2544661 55.69371839

43.76933274 99.37348265 55.60414991

43.36958596 98.96808255 55.5984966

35.11114267 90.69859962 55.58745694

29.48940031 85.03813193 55.54873162

35.03258269 90.44736221 55.41477952

55.47976701 110.818343 55.33857603

38.04746419 93.34466638 55.29720219

40.13238539 95.37388609 55.2415007

44.19099554 99.42563929 55.23464375

33.24656324 87.76949553 54.52293229

36.154565782 91.34577213 55.19111431

27.2754491 82.27419222 54.99874311

36.59699818 91.44689379 54.84989561

37.48100881 92.20830691 54.727298

19.17808219 73.82692962 54.64884742

21.54074074 76.08572124 54.5449805

45.20486556 99.55872914 54.35386358

32.60195759 86.63771915 54.03576157

28.03761445 82.01902665 53.9814122

30.81305821 84.78776478 53.97470658

41.22497055 95.085443531 53.86046476

42.82815987 96.57675493 53.74859506

52.01193335 105.6743612 53.6624278

43.85063378 97.46473998 53.61410621

39.74584291 93.29913695 53.55329404

41.15745598 94.72268477 53.56522879

−19.8129509 33.73781755 53.55076845

13.28219273 66.62503346 53.34284073

40.42061339 93.71157747 53.29096408

50.38587118 103.6178964 53.23202524

45.37762426 98.52069172 53.14306746

36.25704957 89.38134811 53.12429854

30.70619785 83.66680278 52.96060492

55.56846133 108.4823958 52.91393446

36.80576864 89.70132202 52.89555338

38.82044965 91.59237753 52.77192788

50.81668419 103.4121817 52.59549746

46.42363385 99.00409172 52.58045787

69.98574323 122.2854715 52.29972831

25.26217665 77.54573418 52.28355753

39.96352925 92.21643287 52.25290362

47.00574838 99.25113873 52.24539035

53.4905597 105.6295695 52.13900976

49.44050318 101.5763783 52.13587515

39.83535868 91.73339804 51.89803936

54.02808574 105.7595451 51.73145935

51.20524073 102.9357651 51.73052441

43.22472716 94.84355045 51.61882329

42.5929832 94.16497422 51.57199103

32.55627595 83.96631306 51.41003711

47.31068916 98.28460368 50.97391452

46.55678968 97.50512926 50.94833958

34.10555385 85.00081606 50.89526221

30.59011861 81.34023006 50.75011146

55.74433657 106.4705403 50.72620373

44.76047904 95.3640195 50.60354046

−20.05671367 30.41972806 50.47644174

35.60617193 85.8825454 50.27637346

57.07388105 107.3051263 50.2312452

48.7076967 98.89474361 50.1870469

56.5514328 106.3303342 49.77890139

50.4177415 100.1803601 49.76261658

46.79236779 96.49484536 49.70247757

56.60157162 106.2747212 49.67314961

33.00999412 82.65729799 49.64730387

29.33477056 78.76829966 49.43352911

33.27874504 82.61218664 49.33344159

50.5499649 99.82531364 49.27534875

30.48780488 79.68386424 49.19605936

47.95352324 96.90361633 48.95009309

35.81991695 84.74370394 48.92378699

47.9673093 96.80006481 48.8327555

33.22160149 81.90084605 48.67924456

48.95344507 97.58836088 48.63491581

57.44228744 105.7704113 48.32812386

44.25172198 92.47479302 48.22307104

44.23800949 92.39348471 48.15547522

215.6015433 263.737406 48.13586273

56.33127545 104.4315992 48.10032378

55.14812663 103.2306124 48.08248577

64.63766028 112.7047864 48.0671261

44.40577956 92.47084671 48.06506715

51.18862475 99.22340759 48.03478284

20.34336068 68.23874445 47.89538377

44.3557943 92.23836973 47.88257544

67.95159426 115.7456877 47.79409342

55.76098501 103.5302403 47.76925533

48.75160508 96.45841953 47.70681445

48.26958106 95.97238934 47.70280828

69.86140798 117.5434343 47.68202636

48.52722404 96.19903793 47.67181389 40.64114251 88.27689138 47.63564887

43.06535747 90.61530929 47.54995182

40.42192793 87.79749734 47.3756941

62.7184466 110.0825659 47.36411929

58.50862999 105.8574397 47.34880968

34.93352497 82.24076281 47.30723784

53.71064468 101.0010865 47.29044177

28.42089641 75.68202804 47.26113163

47.02449336 94.14723747 47.12274411

55.0937101 102.2099448 47.11623465

30.48240503 77.54573418 47.06332914

51.13822161 98.19719754 47.0589759

65.41604755 112.4344762 47.01842869

48.322054 95.33022533 47.00817133

35.82692457 82.4672171 46.6402925

45.64428312 92.1013059 46.45702277

49.68408533 96.06979643 46.38571109

48.65415987 95.02703588 46.37287601

58.11789038 104.3981674 46.28027705

40.50995892 86.74650699 46.23654806

44.35757282 90.48873778 46.13116496

47.01730419 93.0593376 46.04203341

63.36025402 109.3342199 45.97396591

40.75591985 86.72661643 45.97069657

66.09053804 111.9999168 45.90937874

69.53461354 115.1985743 45.66396079

56.10428305 101.763264 45.65898096

42.83942002 88.37759042 45.5381704

50.06136852 95.57695446 45.51558595

52.76268116 98.25183684 45.48915568

53.00666405 98.46315619 45.45649214

47.98902391 93.41583758 45.42681367

58.33277892 103.7511722 45.41839332

55.45434348 100.8550361 45.4006926

68.79611916 114.1804937 45.38437457

47.68581216 92.92784163 45.24202948

37.60579356 82.80346821 45.19767465

49.80397458 94.9926722 45.18869762

52.42631325 97.49812983 45.07181658

TABLE 1-O Structure

TABLE 1-P Bio- MCL-1% BCL-XL % active Structure Bound Bound Spec ValuesMyricetin

34.13126397 102.3629331 68.23166909 Quercetin

16.68871081 105.3416001 88.65288929 Tyrphostin 47

34.54623163 100.760843 66.2146114 Manoalide

35.01128141 106.8345939 71.8233125 U73122

56.60157162 106.2747212 49.67314961 Baicailen

10.32290702 104.1661092 93.84320213 Dyclonine

1.259544221 90.57802572 89.3184815 Bithionol

27.20036677 100.4228001 73.2243335 Gossypol

3.955080904 101.0243434 97.0692625 Hexa- chloro- phene

41.01271009 100.3606236 59.34791354 Celastrol

32.90443509 97.09434568 64.18991059 Tannic Acid

15.93536133 98.2573418 82.32198048

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1. A compound of the formula: A)

Wherein: X is ═O or —O—R³; R¹ is substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, —S—R^(2a),—SO₂—R^(2a); R² is hydrogen, halogen, —O—R^(2a), —NH—R^(2a), —S—CN,—S—R^(2a), —S—CH²—R^(2a) or —SO₂—R^(2a); R^(2a) is C₁-C₈ alkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, or substituted orunsubstituted heterocycloalkyl; and R³ is hydrogen, C₁-C₈ alkylsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, or substituted orunsubstituted heterocycloalkyl; or B)

wherein: R⁴ and R⁵ are independently hydrogen, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, or substituted or unsubstitutedheterocycloalkyl or are taken together to form a 5-7 membered,optionally substituted, heteroaryl or heterocycloalkyl, provided that nomore than one of R⁴ and R⁵ is hydrogen; And R⁶ is substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, or substituted or unsubstitutedheterocycloalkyl; or C)

Wherein R⁷ is CH₂—CO—R^(7a); —CH₂—NH—SO₂—R^(7a); and R^(7a) issubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, or substituted orunsubstituted heterocycloalkyl; or D)

Wherein R⁸ is —CO—R^(8a); —CO—NH—(CH₂)_(n)—R^(8a); —CO—CH₂—R^(8a),—NH—CO—NH—R^(8a); —NH—SO₂—NH—R^(8a); or —NH—CO—R^(8a); —NH—SO₂—R^(8a)and R^(8a) is substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl; or E)

wherein R⁹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkeynyl, C₂-C₆ alkynyl,—NH₂, —NHR^(9a), —NR^(9a)R^(9b), —COH, —COR^(9a), —COOH, or —COOR^(9a);R^(9a) and R^(9b) are independently C₁-C₆ alkyl, C₂-C₆ alkeynyl, C₂-C₆alkynyl, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, or substituted orunsubstituted heterocycloalkyl W is —R¹⁰, —CO—R¹⁰, —CO—(CH₂)_(n)—R¹⁰,—CO—NH—R¹⁰, —CO—NH—(CH₂)_(n)—R¹⁰, —CO—NH—CO—NH—(CH₂)_(n)—R¹⁰,—CO—NH—SO—NH—(CH₂)_(n)—R¹⁰, —SO—(CH₂)_(n)—R¹⁰, —SO—NH—(CH₂)_(n)—R¹⁰,—SO—NH—CO—NH—(CH₂)_(n)—R¹⁰, —SO—NH—SO—NH—(CH₂)_(n)—R¹⁰, or the group

wherein * represents the point of attachment; R¹⁰ is Z or substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, or substituted or unsubstitutedheterocycloalkyl; R¹² is Z or hydrogen, C₁-C₆ alkyl or—(CH₂)_(n)—R^(12a), R^(12a) is Z or substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, or substituted or unsubstituted heterocycloalkyl; or F)

wherein R¹³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or C₁-C₆alkoxy, and R¹⁴ is substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl wherein R¹⁴ may be furthersubstituted by Z; or G)

wherein R¹⁵ is hydrogen, hydroxyl, halogen, —CO—(CH₂)_(n)—R^(15a),—CO—NH—(CH₂)_(n)—R^(15a), —CO—NH—CO—NH—(CH₂)_(n)—R^(15a),—CO—NH—SO—NH—(CH₂)_(n)—R^(15a), NH—CO—(CH₂)_(n)—R^(15a),NH—CO—NH—(CH₂)_(n)—R^(15a), —SO—(CH₂)_(n)—R^(15a),—SO—NH—(CH₂)_(n)—R^(15a), —SO—NH—CO—NH—(CH₂)_(n)—R^(15a),—SO—NH—SO—NH—(CH₂)_(n)—R^(15a), or —NH—SO₂—R^(15a); alkyl C₁-C₈—R15aR^(15a) is substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl; and R¹⁶ is hydrogen,halogen, hydroxyl or substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl; or H)

wherein X is O or S; R¹⁷ is substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, or substituted or unsubstituted heterocycloalkyl, whereinR¹⁷ may be further substituted by Z; R¹⁸ is hydrogen, C₁-C₈ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, or C₁-C₆ alkoxy, wherein R¹⁸ may be furthersubstituted by Z; and R¹⁹ is hydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, or C₁-C₆ alkoxy wherein R¹⁹ may be further substituted by Z; orI)

wherein Y¹ is N, NH, CH or CH₂; Y² is O, S, or NH; ---- represents anoptional double bond; R²⁰ is substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, or substituted or unsubstituted heterocycloalkyl, whereinR²⁰ may be further substituted by Z; R²¹ represents ═S, ═NH, —NHR^(21a),—CHR^(21a), ═NR^(21a), or ═CCO—R^(21a); and R^(21a) is substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, or substituted or unsubstitutedheterocycloalkyl, wherein R^(21a) may be further substituted by Z; or J)

wherein R²² is substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl wherein R²² may be furthersubstituted by Z; R²³ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, orC₁-C₆ alkoxy, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl wherein R²³ may be furthersubstituted by Z; and R²⁴ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,or C₁-C₆ alkoxy, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl wherein R²⁴ may be furthersubstituted by Z; or K)

wherein R²⁵ is —CO—R^(25a); —CO—NH—(CH₂)_(n)—R^(25a); —CO—CH₂—R^(25a),—NH—CO—NH—R^(25a); —NH—SO₂—NH—R^(25a); or —NH—CO—R^(25a);—NH—SO₂—R^(25a) R^(25a) is substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, or substituted or unsubstituted heterocycloalkyl wherein R²²may be further substituted by Z; or L)

wherein R²⁶ is substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted heterocycloalkyl wherein R²² may be furthersubstituted by Z; or and wherein in each of formulas A-L: eachoccurrence of n is independently an integer from 0-4; each occurrence ofm is independently an integer from 0-3; each occurrence of p isindependently an integer from 0-2; each occurrence of q is independentlyan integer from 0-1; each occurrence of Z, Z′ and Z″ is independentlyhalogen, hydroxyl, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or C₁-C₆alkoxy, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, or substituted orunsubstituted heterocycloalkyl, or M) a compound having the formulaT¹L-T²_(u)Q-T² wherein: T¹ represents a compound of the formula A-L;each T² independently represents another compound of the formula A-Lwhich may be the same or different from T¹; each Q represents a directbond, a C₁-C₈ alkylene linker, a C₂-C₈ alkenylene linker, a C₂-C₈alkynylene linker, an amide linker, or a sulfonamide linker formed byjoining together a Z, Z′, or Z″ moiety of T¹ with a Z, Z′, or Z″ moietyof each subsequent T²; and u represents an integer from 0-5 or apharmaceutically acceptable salt, solvate, hydrate or stereoisomerthereof.
 2. The compound according to claim 1 having the formula:T¹L-T²_(u)Q-T² wherein: T¹ represents a compound of the formula A-L;each T² independently represents another compound of the formula A-Lwhich may be the same or different from T¹; each Q represents a directbond, a C₁-C₈ alkylene linker, a C₂-C₈ alkenylene linker, or a C₂-C₈alkynylene linker formed by joining together a Z, Z′, or Z″ moiety of T¹with a Z, Z′, or Z″ moiety of each subsequent T²; and u represents aninteger from 0-5 or a pharmaceutically acceptable salt, solvate, hydrateor stereoisomer thereof.
 3. The compound according to claim 2, wherein uis 0 and Q is a direct bond.
 4. The compound according to claim 2,wherein u is 0 and Q is a C₁-C₈ alkylene linker.
 5. The compoundaccording to claim 2, wherein u is 1 and each Q is independently adirect bond or a C₁-C₈ alkylene linker.
 6. A pharmaceutical compositioncomprising a compound according to claim 1 or a physiologicallyacceptable salt, solvate, hydrate or stereoisomer thereof and apharmaceutically acceptable diluent or carrier.
 7. The pharmaceuticalcomposition of claim 6 wherein the compound is present in atherapeutically effective amount.
 8. The pharmaceutical composition ofclaim 6 further comprising at least one further active compound.
 9. Thepharmaceutical composition of claim 8, wherein the further activecompound is a death receptor stimulant, an HDAC inhibitor, a proteasomeinhibitor, a BCL-2 family inhibitor, a kinase inhibitor, a mitoticinhibitor, a nucleoside analog, an anti-cancer monoclonal antibody, acorticosteroid, a DNA-damaging agent, or an antimetabolite
 10. Apackaged pharmaceutical composition comprising a container, thepharmaceutical composition of claim 6 and instructions for using thepharmaceutical composition to treat a disease or condition in a mammal.11. A method of selectively modulating MCL-1 in cells comprisingcontacting a cell with one or more compounds of claim 1, therebyregulating MCL-1 in said cell.
 12. A method for modulating apoptoticcell death in a cell comprising contacting a cell with one or morecompounds of claim 1, thereby regulating apoptotic cell death in saidcell.
 13. A method for modulating autophagy in a cell comprisingcontacting a cell with one or more compounds of claim 1, therebyregulating autophagy in said cell.
 14. A method for modulating necroticcell death in a cell comprising contacting a cell with one or morecompounds of claim 1, thereby regulating necrotic cell death in saidcell.
 15. A method for modulating metabolism in a cell comprisingcontacting a cell with one or more compounds of claim 1, therebyregulating energy production and consumption to effect cell viability insaid cell.
 16. A method for modulating cell division in a cellcomprising contacting a cell with one or more compounds of claim 1,thereby regulating proliferation in said cell. 17.-19. (canceled)
 20. Amethod for modulating multimerization of MCL-1 in a cell comprisingcontacting said cell with one or more compounds of claim 1, therebyregulating multimerization of MCL-1 in said cell.
 21. A method oftreating a hyperproliferative disorder in a mammal comprisingadministering to a mammal in need thereof, a therapeutically effectiveamount of one or more compounds of claim
 1. 22. The method of claim 14wherein the hyperproliferative disorder is cancer.
 23. The method ofclaim 15 wherein the hyperproliferative disorder is refractory cancer.24.-36. (canceled)